Stabilised Compositions of Factor VII Polypeptides

ABSTRACT

The invention relates to chemically as well as physically stable kits and compositions comprising polypeptides, in particular Factor VII or Factor VII-related polypeptides, such that these compositions can be stored, handled and used at room temperature.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/407,266, filed Mar. 19, 2009, which is a continuation of U.S. patentapplication Ser. No. 11/450,783, filed Jun. 9, 2006 (Abandoned), whichis a continuation of International Patent Application No.PCT/EP2004/053587, filed Dec. 17, 2004, which claims priority fromDanish Patent Application No. PA 2003 01901, filed Dec. 19, 2003; and toU.S. Patent Application No. 60/531,728, filed Dec. 22, 2003.

FIELD OF INVENTION

The present invention relates to kits comprising chemically as well asphysically stable compositions comprising Factor VII or a FactorVII-related polypeptide such that these compositions can be stored,handled and used at room temperature.

BACKGROUND OF THE INVENTION

Medicaments containing polypeptides are complex compositions. Whendeveloping such a medicament several parameters need to be considered.By example, the medicament needs to be effective, safe and lead to goodpatient compliance. Moreover, the medicament may be formulated forparenteral administration using pharmaceutically acceptable excipients,which will have to meet with the approval of various world-wide medicalregulatory agencies. For the purpose of parenteral administration, it ishighly desirable that the formulation is approximately isotonic and thatthe pH of the formulation is in a physiologically suitable range uponinjection/infusion, otherwise it may result in pain and discomfort forthe patient. For a general review of polypeptide formulations, see, forexample, Cleland et al.: The development of stable protein formulations:A closer look at protein aggregation, deamidation and oxidation,Critical Reviews in Therapeutic Drug Carrier Systems 1993, 10(4):307-377; and Wang et al., Parenteral formulations of polypeptides andpeptides: Stability and stabilizers, Journal of Parenteral Science andTechnology 1988 (Supplement), 42 (2S).

However, for medicaments comprising polypeptides the safety may directlybe related to the physical and chemical stability of the polypeptide.Polypeptides are susceptible to physical degradation, includingdenaturation and aggregation such as the formation of soluble orinsoluble aggregates in the form of dimers, oligomers and polymers, orto chemical degradation, including for example, hydrolysis, deamidationand oxidation. Consequently, the said physical and chemical instabilitymay lead to loss of activity of the polypeptide, formation of toxic andimmunogenic degradation products, in case of coagulation factorpolypeptides there is serious risk of introducing thrombosis uponinjection of the degraded polypeptides, clogging of needles used forinjections and risk of non-homogeneity, to name just a few.

Thus, compositions comprising polypeptides need to be stabilised so asallowing storage and handling at ambient temperatures. One approach ofstabilising a polypeptide relates to removal of water from thepolypeptide, e.g. such as providing the polypeptide in the form of alyophilised cake, the final matter obtained in a freeze-drying process.However, the freeze-drying process itself is also harmful topolypeptides; during freeze-drying, the polypeptide solution is firstcooled until adequately frozen and bulk water in the polypeptidesolution will form ice at this stage. The polypeptide is hereby prone tofreeze-induced stress resulting in deformation and precipitation. In thenext step, the so-called primary drying stage, the ice sublimes and inthe secondary drying stage, adsorbed or bound water is removed underelevated temperatures. During this water removal, the polypeptides mayloose their proper conformation that is provided mainly through hydrogenbonding.

Therefore, to preserve polypeptide conformation, activity and stabilityduring freeze-drying, the polypeptide solution needs to be supplementedwith sufficient amounts of proper excipients with cryoprotectant and/orlyoprotectant properties so as to protect the polypeptide fromfreeze-induced stress and/or stress during removal of water,respectively.

U.S. 20010031721 A1 (American Home Products) concerns highlyconcentrated, lyophilised, and liquid Factor IX formulations.

WO 97/26909 (Genetics Institute) concerns lyophilised preparations ofFactor IX suitable for storage and administration. The preparations maycomprise sucrose or mannitol as a cryoprotectant.

WO 95/28954 (Genetics Institute) concerns preparations of Factor IXsuitable for storage and administration. The preparations may comprisesucrose as a cryoprotectant.

Additionally, when providing a lyophilised product, an essential featurerelates to the properties of the lyophilised cake. It needs to have goodproperties as to its form and structure, i.e. it should not collapse inthat such collapsed cakes can be hard or even impossible to dissolve(reconstitute) before use. Conversely, the physical structure of thelyophilised cake may not be too loosen and soft. Therefore, one or moreso-called bulking agents are added to the polypeptide solution beforefreeze-drying.

Apart from choosing the right bulking agents it is also essential toavoid excipients which destabilises the physical properties of the cake.The concentration of these substances should be as low as possible.Furthermore, it is important that the reconstituted solution is not toohypotonic or hypertonic as this will cause injection inconvenience oreven pain for the patient when administered. Therefore, it is normallynecessary to add tonicity to the composition. Another excipient could bea buffer substance in order to keep the pH of the reconstituted solutionstable during storage.

Vitamin K-dependent polypeptides are a group of polypeptides involved inthe blood clotting process; the group include factor VII, factor IX,factor X, factor II, Protein C, Protein S, gas6, and bone matrix Glapolypeptide or can be a protease selected from the group consisting offactor VIIa, factor IXa, factor Xa, factor IIa, and activated protein C.Factors VIIa, IXa, and Xa are particularly useful proteases. Factor VIIIis a polypeptides involved in the blood clotting process. It can be madeby recombinant techniques or prepared from plasma and is widely used intreatment of bleeding episodes in haemophilia patients.

Factor VII is a polypeptide involved in the blood clotting process.Today, Factor VIIa can be made by recombinant techniques (rFVIIa) and iswidely used as a pro-haemostatic agent. Factor VII (human wild-type) hasbeen described in U.S. Pat. No. 4,784,950. rFVIIa offers today a rapidand highly effective pro-haemostatic response in haemophilic individualsexperiencing bleeding. Advantageously, rFVIIa can be used for treatinghaemophilic individuals that cannot be treated with other coagulationfactor products due to antibody formation. Also individuals sufferingfrom Factor VII deficiency or individuals having a normal coagulationsystem but still experiencing excessive bleeding can be treatedsuccessfully with rFVIIa.

Today, recombinantly-made FVII polypeptide is provided as freeze-driedproduct that is meant to be stored at temperatures between about 2 andabout 8° C. The requirement of cooled conditions causes a burden to andis inconvenient for the manufacturer or provider as well as the end user(the patient).

The actual recombinantly-made FVII product is NovoSeven® (Novo NordiskA/S, Denmark) that consists of 1.2 mg recombinant human Factor VIIa,5.84 mg NaCl, 2.94 mg CaCl2, 2 H2O, 2.64 mg Glycylglycine, 0.14 mgpolysorbate 80 and 60.0 mg mannitol. When reconstituted by 2.0 ml ofwater for injection (WFI), the pH is 5.5 and the thus preparedFVII-containing solution is sufficiently stable for 24 hours at roomtemperature.

The present investigators have found that upon storage of thelyophilised NovoSeven® product for 6 months at 25° C. about 6 to 7% w/wof the initial content of the rFVIIa is present in the form ofaggregates.

Thus, compositions comprising Factor VII polypeptides need to bestabilised so as allowing storage and handling at ambient temperatures.

It is an objective of the present invention to provide improvedcompositions, kits, and methods for producing these, wherein the drycompositions comprising the polypeptides are stabilized against chemicaland physical degradation (such as, e.g., forming less dimer/oligomerdegradation forms); with good properties of the lyophilised cake as toits form and structure, i.e. it should not collapse; with good andstable physical structure of the lyophilised cake; where the drycomposition is devoid of excipients which destabilises the physicalproperties of the cake, e.g., by decreasing the eutectic melting pointand thus increasing the risk of collapse of the cake; wherein thereconstituted composition prepared by dissolving the drypolypeptide-containing composition in the administration vehicle isisotonic, or closely isotonic, and has a well-defined pH (pH-stable).Particularly, it is an object to provide improved compositionscomprising Factor VII polypeptides, substantially without the presenceof degradation products and without decreased activity of the Factor VIIpolypeptides, preferable after prolonged storage at ambient conditions,e.g. for at least 6 months. Furthermore, it is an objective that thestable compositions are suitable for parenteral administration so as notto cause any inconvenience for the patient.

SUMMARY OF THE INVENTION

It has been found by the present investigators thatpolypeptide-containing medicaments can be provided as a kit of partscomprising a first unit form consisting of a dry (e.g., a freeze-dried)composition comprising a polypeptide and at least one stabilizing agentwherein the composition has a moisture content of not more than about3%, and container means for containing said first unit form; and, incontainer means for containing such a unit, a second unit formconsisting of an administration vehicle comprising a solvent forsolution (reconstitution) of said composition and at least one of thecomponents selected from the list of: (i) an agent suitable for keepingthe pH of said composition in the range of 3 to 9 when dissolved inaqueous solvent in an amount of from about 0.1 mM to 100 mM; and (ii) atonicity modifying agent in an amount sufficient to make essentiallyisotonic the reconstituted solution resulting from dissolving thecomposition of the first unit form in the administration vehicle of thesecond unit form.

Substances which usually are present in the formulation like buffersubstances and tonicity modifiers will very often decrease the eutecticmelting point and will increase the risk of collapse of the cake. Ifthese substances are present during freeze drying the temperature of theice during the primary drying must be lowered to avoid collapse andconsequently the time for freeze drying is prolonged. The concentrationof these substances in the freeze-dried cake should be as low aspossible or they should be completely avoided. Instead they maybeneficially be added to the reconstitution liquid.

By lowering the concentration of these excipients or completely removingthem, the reconstituted solution will in some instances become hypotonicand it is necessary the add tonicity modifiers to the solvent so as toobtain a solution with the needed tonicity, such as isotonicity, orclosely so (“essentially isotonic”). Another necessary excipient in thesolvent could be a buffer substance in order to keep the pH of thereconstituted solution stable during storage.

The kit of parts is sufficiently stable so as to allow for storage atroom temperature for about at least 8 months.

Furthermore, it has been found that Factor VII polypeptides can beprovided in a composition that is sufficient stable so as to allow forstorage at room temperature for about at least 8 months. Theinvestigators have found that the stabilisation relates to the propercombining of some pharmaceutically acceptable excipients.

Accordingly, the present invention relates in a first aspect to a kit(containing a pharmaceutical medicament/treatment), said kit comprising

-   a) a composition comprising a polypeptide and at least one    stabilizing agent, wherein the composition has a moisture content of    not more than about 3%, in a first unit form, and container means    for containing said first unit form; and-   b) an administration vehicle comprising a solvent for reconstitution    (solution) of said composition and at least one of the components    selected from the list of:    -   (i) an agent suitable for keeping the pH of said composition in        the range of 3 to 9 when dissolved in aqueous solvent, wherein        the agent is present in an amount of from about 0.1 mM to 100        mM,    -   (ii) a tonicity modifying agent in an amount sufficient to make        the reconstituted solution resulting from dissolving the        composition of the first unit form in the administration vehicle        of the second unit form essentially isotonic;-   in a second unit form, and container means for containing said    second unit form.

In a second aspect, the invention relates to a method for preparing aliquid formulation of a polypeptide, the method comprising the steps of:

-   a) providing a first and a second unit form as described above, and-   b) mixing said first and second unit forms so as to provide a    dissolved liquid solution of the composition in the administration    vehicle.

In a third and fourth aspect, the invention relates to a method fortreating a coagulation factor-responsive syndrome, comprisingadministering to a subject in need thereof an effective amount of aliquid formulation of a coagulation factor prepared by the methoddescribed above, and to the use of said formulation for the preparationof a medicament in the form of a kit as defined above for treatment of aFactor VII-responsive syndrome.

In a fifth aspect, the invention relates to a composition comprising aFactor VII polypeptide, and at least one stabilizing agent selected fromthe group consisting of

-   a) a combination of an antioxidant and mannitol;-   b) a combination of methionine and a polyol;-   c) a combination of a saccharide and mannitol;-   d) a combination of sucrose and a polyol;-   e) methionine; and-   a polysorbate surfactant in an amount of from about 0.06 to 0.08    mg/mL;-   said composition having a moisture content of not more than about    3%;

In a sixth aspect, the invention relates to a method of preparing theabove defined compositions, comprising the steps of:

-   i) providing a Factor VII polypeptide in a solution comprising at    least one stabilizing agent selected from the group consisting of-   a) a combination of an antioxidant and mannitol;-   b) a combination of methionine and a polyol;-   c) a combination of a saccharide and mannitol;-   d) a combination of sucrose and a polyol; and-   e) methionine; and-   a polysorbate surfactant in an amount of from about 0.06 to 0.08    mg/mL;-   ii) processing said solution so as to obtain a solid composition    with a moisture content not more than about 3% w/w.

In a seventh and eighth aspect, the invention relates to a method fortreating a FVII-responsive syndrome, comprising administering to asubject in need thereof an effective amount of a composition as definedabove, and to the use of Factor VII polypeptide for the preparation of amedicament for treating a Factor VII-responsive syndrome, saidmedicament comprising a composition as defined above.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to storage-stable kits and compositionscomprising polypeptides, including FVIII polypeptides, VitaminK-dependent polypeptides, and FVII polypeptides. The compositions can bestored at room temperature for an extended period of time withoutcausing substantial degradation of the polypeptide. By room temperatureis meant the ambient temperature inside a room; it normally ranges fromabout 5° C. to about 40° C., such as from about 10° C. to 30° C., or 15°C. to 25° C.

By proper predetermined combination of particular pharmaceuticallyacceptable excipients, the present investigators have providedstabilised compositions comprising polypeptides, particularly Factor VIIpolypeptides, thus allowing the compositions to be stored at roomtemperature for an extended period of time such as at least about 8months. Advantageously, the stabilised compositions need not to bestored at cooled conditions, such as between 2 and 8° C.

The present invention also concerns storage-stable compositions that arestable for at least about 8 months upon storage at about 30° C. Thecomposition is preferably stored in the dark. Thus, the presentinvention makes it possible to store such compositions at roomtemperature without increasing the risk of adverse events to the patientadministering such compositions. Advantageously, the improvedstorage-stability will also result in reduced cost in that no specialcooled conditions are required upon storage, further resulting in moreconvenient handling of the composition by the user.

Polypeptides to be formulated in accordance with the present inventionincludes, without limitation, blood coagulation factors includingvitamin K-dependent polypeptides, such as, e.g., without limitation,factor VIII, factor V, factor XI, factor VII, factor IX, factor X,factor II, Protein C, Protein S, gas6, and bone matrix Gla polypeptide;activated FVIII, factor Va, factor XIa, factor VIIa, factor IXa, factorXa, factor IIa, and activated Protein C.

The term “Vitamin K-dependent polypeptide” includes polypeptidesselected from the group consisting of factor VII, factor IX, factor X,factor II, Protein C, Protein S, gas6, and bone matrix Gla polypeptideor can be a protease selected from the group consisting of factor VIIa,factor IXa, factor Xa, factor IIa, and activated Protein C. FactorsVIIa, IXa, and Xa are particularly useful proteases.

The term “Factor VII polypeptide” is denoted to mean any Factor VIIpolypeptide that is effective in preventing or treating bleeding. Thisincludes Factor VII polypeptides derived from blood or plasma, orproduced by recombinant means.

As used herein, “Factor VII polypeptide” encompasses, withoutlimitation, Factor VII, including variants thereof, as well as FactorVII-related polypeptides, Factor VII derivatives and Factor VIIconjugates. The term “Factor VII” is intended to encompass, withoutlimitation, polypeptides having the amino acid sequence 1-406 ofwild-type human Factor VII (as disclosed in U.S. Pat. No. 4,784,950), aswell as wild-type Factor VII derived from other species, such as, e.g.,bovine, porcine, canine, murine, and salmon, said Factor VII derivedfrom blood or plasma, or produced by recombinant means. It furtherencompasses natural allelic variations of Factor VII that may exist andoccur from one individual to another. Also, the degree and location ofglycosylation or other post-translation modifications may vary dependingon the chosen host cells and the nature of the host cellularenvironment. The term “Factor VII” is also intended to encompass FactorVII polypeptides in their uncleaved (zymogen) form, as well as thosethat have been proteolytically processed to yield their respectivebioactive forms, which may be designated Factor VIIa. Typically, FactorVII is cleaved between residues 152 and 153 to yield Factor VIIa.

The term “Factor VII derivative” as used herein, is intended todesignate a FVII polypeptide exhibiting substantially the same orimproved biological activity relative to wild-type Factor VII, in whichone or more of the amino acids of the parent peptide have beengenetically and/or chemically and/or enzymatically modified, e.g. byalkylation, glycosylation, PEGylation, acylation, ester formation oramide formation or the like. This includes but is not limited toPEGylated human Factor VIIa, cysteine-PEGylated human Factor VIIa andvariants thereof. Non-limiting examples of Factor VII derivativesincludes GlycoPegylated FVII derivatives as disclosed in WO 03/31464 andUS Patent applications US 20040043446, US 20040063911, US 20040142856,US 20040137557, and US 20040132640 (Neose Technologies, Inc.); FVIIconjugates as disclosed in WO 01/04287, US patent application20030165996, WO 01/58935, WO 03/93465 (Maxygen ApS) and WO 02/02764, USpatent application 20030211094 (University of Minnesota).

The term “improved biological activity” refers to FVII polypeptides withi) substantially the same or increased proteolytic activity compared torecombinant wild type human Factor VIIa or ii) to FVII polypeptides withsubstantially the same or increased TF binding activity compared torecombinant wild type human Factor VIIa or iii) to FVII polypeptideswith substantially the same or increased half life in blood plasmacompared to recombinant wild type human Factor VIIa. The term “PEGylatedhuman Factor VIIa” means human Factor VIIa, having a PEG moleculeconjugated to a human Factor VIIa polypeptide. It is to be understood,that the PEG molecule may be attached to any part of the Factor VIIapolypeptide including any amino acid residue or carbohydrate moiety ofthe Factor VIIa polypeptide. The term “cysteine-PEGylated human FactorVIIa” means Factor VIIa having a PEG molecule conjugated to a sulfhydrylgroup of a cysteine introduced in human Factor VIIa.

As mentioned, the term “Factor VII polypeptides” is also denoted to mean“Factor VII-related polypeptides” The term “Factor VII-relatedpolypeptides” are intended to encompass such polypeptides in theiruncleaved (zymogen) form, as well as those that have beenproteolytically processed to yield their respective bioactive forms. Asused herein, “Factor VII-related polypeptides” encompass, withoutlimitation, polypeptides exhibiting substantially the same or improvedbiological activity relative to wild-type human Factor VII andpolypeptides wherein the biological activity has been substantiallyreduced relative to the activity of wild-type human factor VIIa (asdisclosed in U.S. Pat. No. 4,784,950). These polypeptides include,without limitation, Factor VII or Factor VIIa that has been chemicallymodified, such as, e.g., by reacting factor VII with an irreversibleinhibitor such as an organophosphor compound, a sulfonyl fluoride, apeptide halomethyl ketone or an azapeptide, or by acylation, bynon-limiting example, and Factor VII variants into which specific aminoacid sequence alterations have been introduced that slightly modify orimprove the biological activity of the polypeptide, such as, e.g.,polypeptides wherein the catalytic activity of factor VIIa is inhibitedby chemical derivatization of the catalytic site, or triad.

The term “catalytic site” or “active site”, when used herein withreference to FVIIa, refer to the catalytic and zymogen substrate bindingsite, including the “S₁” site of FVIIa as that term is defined bySchecter, I. and Berger, A., (1967) Biochem. Biophys. Res. Commun.7:157-162. The catalytic site of human and bovine Factor VII proteinscomprises the amino acids Ser344, Asp242, and His193 (subscriptnumbering indicating position in the sequence) that are forming aso-called catalytic “triad”. The catalytic sites in Factor VII fromother mammalian species may be determined using presently availabletechniques including, among others, protein isolation and amino acidsequence analysis. Catalytic sites may also be determined by aligning asequence with the sequence of other serine proteases, particularlychymotrypsin, whose active site has been previously determined (Sigleret al., J. Mol. Biol., 35:143-164 (1968)) and therefrom determining fromsaid alignment the analogous active site residues.

Factor VII-related polypeptides, including variants, havingsubstantially the same or improved biological activity relative towild-type Factor VIIa encompass those that exhibit at least about 25%,preferably at least about 50%, more preferably at least about 75%, morepreferably at least about 100%, more preferably at least about 110%,more preferably at least about 120%, and most preferably at least about130% of the specific activity of wild-type Factor VIIa that has beenproduced in the same cell type, when tested in one or more of a clottingassay, proteolysis assay, or TF binding assay as described in thepresent specification.

Factor VII-related polypeptides, including variants, wherein thebiological activity has been substantially reduced relative to theactivity of wild-type human factor VIIa encompass those polypeptidesthat exhibit less than about 25%, more preferably less than about 10%,or 5%, or 3%, or 2%, and most preferably less than about 1% of thespecific activity of wild-type factor VIIa, when tested in one or moreof a clotting assay, FIXa or FXa generation assay, amidolysis orproteolysis assay as described within the present specification

In some embodiments the Factor VII polypeptides are Factor VII-relatedpolypeptides, in particular variants, wherein the ratio between theactivity of said Factor VII polypeptide and the activity of native humanFactor VIIa (wild-type FVIIa) is at least about 1.25 when tested in the“In Vitro Hydrolysis Assay” (see Examples, General Methods, below); inother embodiments, the ratio is at least about 2.0; in furtherembodiments, the ratio is at least about 4.0. In some embodiments of theinvention, the Factor VII polypeptides are Factor VII-relatedpolypeptides, in particular variants, wherein the ratio between theactivity of said Factor VII polypeptide and the activity of native humanFactor VIIa (wild-type FVIIa) is at least about 1.25 when tested in the“In Vitro Proteolysis Assay” (see Examples, General Methods, below); inother embodiments, the ratio is at least about 2.0; in furtherembodiments, the ratio is at least about 4.0; in further embodiments,the ratio is at least about 8.0.

Non-limiting examples of Factor VII variants having substantially thesame or improved biological activity as wild-type Factor VII includeS52A-FVII, S60A-FVII (Lino et al., Arch. Biochem. Biophys. 352: 182-192,1998); L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII,F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII,M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII,V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII,V158D/E296V/M298Q/L305V/K337A-FVII, K157A-FVII, E296V-FVII,E296V/M298Q-FVII, V158D/E296V-FVII, V158D/M298K-FVII, and S336G-FVII;FVIIa variants exhibiting increased proteolytic stability as disclosedin U.S. Pat. No. 5,580,560; Factor VIIa that has been proteolyticallycleaved between residues 290 and 291 or between residues 315 and 316(Mollerup et al., Biotechnol. Bioeng. 48:501-505, 1995); oxidized formsof Factor VIIa (Kornfelt et al., Arch. Biochem. Biophys. 363:43-54,1999); FVII variants as disclosed in PCT/DK02/00189 (corresponding to WO02/077218); and FVII variants exhibiting increased proteolytic stabilityas disclosed in WO 02/38162 (Scripps Research Institute); FVII variantshaving a modified Gla-domain and exhibiting an enhanced membrane bindingas disclosed in WO 99/20767, US patents U.S. Pat. No. 6,017,882 and U.S.Pat. No. 6,747,003, US patent application 20030100506 (University ofMinnesota) and WO 00/66753, US patent applications US 20010018414, US2004220106, and US 200131005, US patents U.S. Pat. No. 6,762,286 andU.S. Pat. No. 6,693,075 (University of Minnesota); and FVII variants asdisclosed in WO 01/58935, US patent U.S. Pat. No. 6,806,063, US patentapplication 20030096338 (Maxygen ApS), WO 03/93465 (Maxygen ApS) and WO04/029091 (Maxygen ApS); FVII variants having increased biologicalactivity compared to wild-type FVIIa as disclosed in WO 01/83725, WO02/22776, WO 02/077218, PCT/DK02/00635, Danish patent application PA2002 01423, Danish patent application PA 2001 01627; WO 02/38162(Scripps Research Institute); and FVIIa variants with enhanced activityas disclosed in JP 2001061479 (Chemo-Sero-Therapeutic Res Inst.).

Non-limiting examples of factor VII polypeptides having substantiallyreduced biological activity relative to wild-type factor VII includeR152E-FVIIa (Wildgoose et al., Biochem 29:3413-3420, 1990), S344A-FVIIa(Kazama et al., J. Biol. Chem. 270:66-72, 1995), FFR-FVIIa (Hoist etal., Eur. J. Vasc. Endovasc. Surg. 15:515-520, 1998), and factor VIIalacking the Gla domain, (Nicolaisen et al., FEBS Letts. 317:245-249,1993). Non-limiting examples also include human FVIIa, which has thelysine residue in position 341 replaced by another amino acid residue;human FVIIa, which has the serine residue in position 344 replaced byanother amino acid residue; human FVIIa, which has the aspartic acidresidue in position 242 replaced by another amino acid residue; humanFVIIa, which has the histidine residue in position 193 replaced byanother amino acid residue; FVII-(K341A); FVII-(S344A); FVII-(D242A);FVII-(H193A); Phe-Phe-Arg-FVII (FFR-FVII), D-Phe-Phe-Arg-FVII(D-FFR-FVII), Phe-Pro-Arg-FVII (FPR-FVII), D-Phe-Pro-Arg-FVII(D-FPR-FVII), L-Glu-Gly-Arg-FVII (EGR-FVII) and D-Glu-Gly-Arg-FVII(D-EGR-FVII), Dansyl-Phe-Phe-Arg-FVII, Dansyl-D-Phe-Phe-Arg-FVII,Dansyl-Phe-Pro-Arg-FVII, Dansyl-D-Phe-Pro-Arg-FVII,Dansyl-L-Glu-Gly-Arg-FVII and Dansyl-D-Glu-Gly-Arg-FVII. Non-limitingexamples of chemically modified factor VII polypeptides and sequencevariants are described, e.g., in U.S. Pat. No. 5,997,864.

Examples of factor VII or factor VII-related polypeptides include,without limitation, wild-type Factor VII, L305V-FVII,L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII,V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII,V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII,V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII,K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII,V158D/M298K-FVII, and S336G-FVII, L305V/K337A-FVII, L305V/V158D-FVII,L305V/E296V-FVII, L305V/M298Q-FVII, L305V/V158T-FVII,L305V/K337A/V158T-FVII, L305V/K337A/M298Q-FVII, L305V/K337A/E296V-FVII,L305V/K337A/V158D-FVII, L305V/V158D/M298Q-FVII, L305V/V158D/E296V-FVII,L305V/V158T/M298Q-FVII, L305V/V158T/E296V-FVII, L305V/E296V/M298Q-FVII,L305V/V158D/E296V/M298Q-FVII, L305V/V158T/E296V/M298Q-FVII,L305V/V158T/K337A/M298Q-FVII, L305V/V158T/E296V/K337A-FVII,L305V/V158D/K337A/M298Q-FVII, L305V/V158D/E296V/K337A-FVII,L305V/V158D/E296V/M298Q/K337A-FVII, L305V/V158T/E296V/M298Q/K337A-FVII,S314E/K316H-FVII, S314E/K316Q-FVII, S314E/L305V-FVII, S314E/K337A-FVII,S314E/V158D-FVII, S314E/E296V-FVII, S314E/M298Q-FVII, S314E/V158T-FVII,K316H/L305V-FVII, K316H/K337A-FVII, K316H/V158D-FVII, K316H/E296V-FVII,K316H/M298Q-FVII, K316H/V158T-FVII, K316Q/L305V-FVII, K316Q/K337A-FVII,K316Q/V158D-FVII, K316Q/E296V-FVII, K316Q/M298Q-FVII, K316Q/V158T-FVII,S314E/L305V/K337A-FVII, S314E/L305V/V158D-FVII, S314E/L305V/E296V-FVII,S314E/L305V/M298Q-FVII, S314E/L305V/V158T-FVII,S314E/L305V/K337A/V158T-FVII, S314E/L305V/K337A/M298Q-FVII,S314E/L305V/K337A/E296V-FVII, S314E/L305V/K337A/V158D-FVII,S314E/L305V/V158D/M298Q-FVII, S314E/L305V/V158D/E296V-FVII,S314E/L305V/V158T/M298Q-FVII, S314E/L305V/V158T/E296V-FVII,S314E/L305V/E296V/M298Q-FVII, S314E/L305V/V158D/E296V/M298Q-FVII,S314E/L305V/V158T/E296V/M298Q-FVII, S314E/L305V/V158T/K337A/M298Q-FVII,S314E/L305V/V158T/E296V/K337A-FVII, S314E/L305V/V158D/K337A/M298Q-FVII,S314E/L305V/V158D/E296V/K337A-FVII,S314E/L305V/V158D/E296V/M298Q/K337A-FVII,S314E/L305V/V158T/E296V/M298Q/K337A-FVII, K316H/L305V/K337A-FVII,K316H/L305V/V158D-FVII, K316H/L305V/E296V-FVII, K316H/L305V/M298Q-FVII,K316H/L305V/V158T-FVII, K316H/L305V/K337A/V158T-FVII,K316H/L305V/K337A/M298Q-FVII, K316H/L305V/K337A/E296V-FVII,K316H/L305V/K337A/V158D-FVII, K316H/L305V/V158D/M298Q-FVII,K316H/L305V/V158D/E296V-FVII, K316H/L305V/V158T/M298Q-FVII,K316H/L305V/V158T/E296V-FVII, K316H/L305V/E296V/M298Q-FVII,K316H/L305V/V158D/E296V/M298Q-FVII, K316H/L305V/V158T/E296V/M298Q-FVII,K316H/L305V/V158T/K337A/M298Q-FVII, K316H/L305V/V158T/E296V/K337A-FVII,K316H/L305V/V158D/K337A/M298Q-FVII, K316H/L305V/V158D/E296V/K337A-FVII,K316H/L305V/V158D/E296V/M298Q/K337A-FVII,K316H/L305V/V158T/E296V/M298Q/K337A-FVII, K316Q/L305V/K337A-FVII,K316Q/L305V/V158D-FVII, K316Q/L305V/E296V-FVII, K316Q/L305V/M298Q-FVII,K316Q/L305V/V158T-FVII, K316Q/L305V/K337A/V158T-FVII,K316Q/L305V/K337A/M298Q-FVII, K316Q/L305V/K337A/E296V-FVII,K316Q/L305V/K337A/V158D-FVII, K316Q/L305V/V158D/M298Q-FVII,K316Q/L305V/V158D/E296V-FVII, K316Q/L305V/V158T/M298Q-FVII,K316Q/L305V/V158T/E296V-FVII, K316Q/L305V/E296V/M298Q-FVII,K316Q/L305V/V158D/E296V/M298Q-FVII, K316Q/L305V/V158T/E296V/M298Q-FVII,K316Q/L305V/V158T/K337A/M298Q-FVII, K316Q/L305V/V158T/E296V/K337A-FVII,K316Q/L305V/V158D/K337A/M298Q-FVII, K316Q/L305V/V158D/E296V/K337A-FVII,K316Q/L305V/V158D/E296V/M298Q/K337A-FVII,K316Q/L305V/V158T/E296V/M298Q/K337A-FVII, F374Y/K337A-FVII,F374Y/V158D-FVII, F374Y/E296V-FVII, F374Y/M298Q-FVII, F374Y/V158T-FVII,F374Y/S314E-FVII, F374Y/L305V-FVII, F374Y/L305V/K337A-FVII,F374Y/L305V/V158D-FVII, F374Y/L305V/E296V-FVII, F374Y/L305V/M298Q-FVII,F374Y/L305V/V158T-FVII, F374Y/L305V/S314E-FVII, F374Y/K337A/S314E-FVII,F374Y/K337A/V158T-FVII, F374Y/K337A/M298Q-FVII, F374Y/K337A/E296V-FVII,F374Y/K337A/V158D-FVII, F374Y/V158D/S314E-FVII, F374Y/V158D/M298Q-FVII,F374Y/V158D/E296V-FVII, F374Y/V158T/S314E-FVII, F374Y/V158T/M298Q-FVII,F374Y/V158T/E296V-FVII, F374Y/E296V/S314E-FVII, F374Y/S314E/M298Q-FVII,F374Y/E296V/M298Q-FVII, F374Y/L305V/K337A/V158D-FVII,F374Y/L305V/K337A/E296V-FVII, F374Y/L305V/K337A/M298Q-FVII,F374Y/L305V/K337A/V158T-FVII, F374Y/L305V/K337A/S314E-FVII,F374Y/L305V/V158D/E296V-FVII, F374Y/L305V/V158D/M298Q-FVII,F374Y/L305V/V158D/S314E-FVII, F374Y/L305V/E296V/M298Q-FVII,F374Y/L305V/E296V/V158T-FVII, F374Y/L305V/E296V/S314E-FVII,F374Y/L305V/M298Q/V158T-FVII, F374Y/L305V/M298Q/S314E-FVII,F374Y/L305V/V158T/S314E-FVII, F374Y/K337A/S314E/V158T-FVII,F374Y/K337A/S314E/M298Q-FVII, F374Y/K337A/S314E/E296V-FVII,F374Y/K337A/S314E/V158D-FVII, F374Y/K337A/V158T/M298Q-FVII,F374Y/K337A/V158T/E296V-FVII, F374Y/K337A/M298Q/E296V-FVII,F374Y/K337A/M298Q/V158D-FVII, F374Y/K337A/E296V/V158D-FVII,F374Y/V158D/S314E/M298Q-FVII, F374Y/V158D/S314E/E296V-FVII,F374Y/V158D/M298Q/E296V-FVII, F374Y/V158T/S314E/E296V-FVII,F374Y/V158T/S314E/M298Q-FVII, F374Y/V158T/M298Q/E296V-FVII,F374Y/E296V/S314E/M298Q-FVII, F374Y/L305V/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/K337A/S314E-FVII, F374Y/E296V/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A-FVII, F374Y/L305V/E296V/M298Q/S314E-FVII,F374Y/V158D/E296V/M298Q/K337A-FVII, F374Y/V158D/E296V/M298Q/S314E-FVII,F374Y/L305V/V158D/K337A/S314E-FVII, F374Y/V158D/M298Q/K337A/S314E-FVII,F374Y/V158D/E296V/K337A/S314E-FVII, F374Y/L305V/V158D/E296V/M298Q-FVII,F374Y/L305V/V158D/M298Q/K337A-FVII, F374Y/L305V/V158D/E296V/K337A-FVII,F374Y/L305V/V158D/M298Q/S314E-FVII, F374Y/L305V/V158D/E296V/S314E-FVII,F374Y/V158T/E296V/M298Q/K337A-FVII, F374Y/V158T/E296V/M298Q/S314E-FVII,F374Y/L305V/V158T/K337A/S314E-FVII, F374Y/V158T/M298Q/K337A/S314E-FVII,F374Y/V158T/E296V/K337A/S314E-FVII, F374Y/L305V/V158T/E296V/M298Q-FVII,F374Y/L305V/V158T/M298Q/K337A-FVII, F374Y/L305V/V158T/E296V/K337A-FVII,F374Y/L305V/V158T/M298Q/S314E-FVII, F374Y/L305V/V158T/E296V/S314E-FVII,F374Y/E296V/M298Q/K337A/V158T/S314E-FVII,F374Y/V158D/E296V/M298Q/K337A/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/S314E-FVII,F374Y/L305V/E296V/M298Q/V158T/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A/V158T-FVII,F374Y/L305V/E296V/K337A/V158T/S314E-FVII,F374Y/L305V/M298Q/K337A/V158T/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/K337A-FVII,F374Y/L305V/V158D/E296V/K337A/S314E-FVII,F374Y/L305V/V158D/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A/V158T/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/K337A/S314E-FVII, S52A-Factor VII,S60A-Factor VII; and P11Q/K33E-FVII, T106N-FVII, K143N/N145T-FVII,V253N-FVII, R290N/A292T-FVII, G291N-FVII, R315N/V317T-FVII,K143N/N145T/R315N/V317T-FVII; FVII having substitutions, additions ordeletions in the amino acid sequence from 233Thr to 240Asn, FVII havingsubstitutions, additions or deletions in the amino acid sequence from304Arg to 329Cys, and FVII having substitutions, deletions, or additionsin the amino acid sequence Ile153-Arg223.

For purposes of the invention, biological activity of Factor VIIpolypeptides (“Factor VII biological activity”) may be quantified bymeasuring the ability of a preparation to promote blood clotting usingFactor VII-deficient plasma and thromboplastin, as described, e.g., inU.S. Pat. No. 5,997,864. In this assay, biological activity is expressedas the reduction in clotting time relative to a control sample and isconverted to “Factor VII units” by comparison with a pooled human serumstandard containing 1 unit/ml Factor VII activity. Alternatively, FactorVIIa biological activity may be quantified by

-   -   (i) Measuring the ability of Factor VIIa or a Factor        VIIa-related polypeptide to produce activated Factor X (Factor        Xa) in a system comprising TF embedded in a lipid membrane and        Factor X. (Persson et al., J. Biol. Chem. 272:19919-19924,        1997);    -   (ii) Measuring Factor X hydrolysis in an aqueous system (“In        Vitro Proteolysis Assay”, see Examples, General Methods, below);    -   (iii) Measuring the physical binding of Factor VIIa or a Factor        V—IIa-related polypeptide to TF using an instrument based on        surface plasmon resonance (Persson, FEBS Letts. 413:359-363,        1997); and    -   (iv) Measuring hydrolysis of a synthetic substrate by Factor        VIIa and/or a Factor VIIa-related polypeptide (“In Vitro        Hydrolysis Assay”, see Examples, General Methods, below); and    -   (v) Measuring generation of thrombin in a TF-independent in        vitro system.

The term “Factor VII biological activity” or “Factor VII activity” isintended to include the ability to generate thrombin; the term alsoincludes the ability to generate thrombin on the surface of activatedplatelets in the absence of tissue Factor.

“Examples, General Methods” of the present specification describes indetail assays useful for assaying FVII biological activity.

Moreover, throughout the present specification, the terms below have thefollowing meaning:

The term “kit” or “kit of parts” is intended to mean a combination of adry product, in a first unit, containing a polypeptide and one or morestabilizing agents; and an administration vehicle consisting of asolvent suitable for dissolving the dry product of the first unit, incombination with at least one buffering agent in an amount of from aboutfrom about 0.1 mM to 100 mM, and/or at least one tonicity modifyingagent, in a second unit. The kit contains a pharmaceutical treatment,particularly of bleeding episodes. Before use, the dry composition ofthe first unit is mixed with and dissolved in the administration vehiclecontained in the second unit, thereby providing a medicament ready foruse.

The medicament ready for use is essentially isotonic.

The term “administration vehicle” is intended to encompasspharmaceutically acceptable, preferably sterile liquids suitable foradministration by injectable means, such as infusion or injection, e.g.,by intravenous, subcutaneous, or intramuscular injection. Theadministration vehicle is preferably aqueous. The administration vehiclecomprises a solvent, or a mixture of solvents, suitable forreconstitution (solution) of the polypeptide composition (e.g., Waterfor Injection/WFI), and one or more agents suitable for keeping the pHof said composition in the range of 3 to 9 when dissolved in aqueoussolvent in an amount of from about 0.1 mM to 100 mM; and/or one or moretonicity modifying agents in an amount sufficient to make essentiallyisotonic the reconstituted solution resulting from dissolving thecomposition of the first unit form in the administration vehicle of thesecond unit form.

The administration vehicle may contain further substances, such as metalsalts, e.g., calcium and/or magnesium salts, amino acids, e.g.,glycylglycine.

The reconstituted compositions are intended for parenteraladministration for prophylactic and/or therapeutic treatment.

An “effective amount” of a polypeptide refers to the amount ofpolypeptide which, when administered in accordance with the invention,produces a measurable improvement in at least one clinical parameter ofhaemostasis known to those of ordinary skill in the art.

It will be understood that an effective amount of a polypeptide may varyaccording to the subject's haemostatic status, which, in turn, may bereflected in one or more clinical parameters, including, e.g., relativelevels of circulating coagulation factors; amount of blood lost; rate ofbleeding; hematocrit, and the like. It will be further understood thatthe single-dose-effective amount may be determined by those of ordinaryskill in the art by routine experimentation, by constructing a matrix ofvalues and testing different points in the matrix.

The term “stabilizing” is intended to encompass minimising the formationof aggregates (insoluble and/or soluble) and/or chemical degradation aswell as providing maintenance of pH and proper conformation of thepolypeptide during storage or production of the compositions so thatsubstantial retention of biological activity and polypeptide stabilityis maintained. Moreover, stabilising is also denoted to meanlyoprotection and cryoprotection of the polypeptide during production ofthe compositions at freeze-drying conditions.

The term “stabilizing agent” is intended to encompass substances, or amixture of substances, that are able to stabilize a polypeptide duringstorage or production of a composition comprising the polypeptide.

The term “structural stabilisation” or “structural stability” isintended to encompass the ability to form a lyophilised plug or cakewith good properties and looks, e.g. such that it does not collapse andis readily dissolved before use.

The term “storage-stable” is intended to define a product that isstabilised upon storage at temperatures between 5° C.-40° C. and remainswithin pre-selected product specifications for a suitable timeperiod—often several months.

The term “physical stability” of Factor VII polypeptides relates to theformation of insoluble and/or soluble aggregates in the form of dimeric,oligomeric and polymeric forms of Factor VII polypeptides as well as anystructural deformation and denaturation of the molecule.

The term “chemical stability” is intended to relate to the formation ofany chemical change in the Factor VII polypeptides upon storage indissolved or solid state at accelerated conditions. By example arehydrolysis, deamidation and oxidation. In particular, thesulphur-containing amino acids are prone to oxidation with the formationof the corresponding sulphoxides.

The term “cryoprotectants” as used herein generally include agents,which provide stability to the polypeptide from freezing-inducedstresses. Examples of cryoprotectants include polyols such as, forexample, mannitol, and include saccharides such as, for example,sucrose, as well as including surfactants such as, for example,polysorbate, poloxamer or polyethylene glycol, and the like.Cryoprotectants also contribute to the tonicity of the formulations.

The term “lyoprotectant” as used herein includes agents that providestability to the polypeptide during water removal upon the dryingprocess of the lyophilisation process. For example by maintaining theproper conformation of the polypeptide. Examples of lyoprotectantsinclude saccharides, in particular di- or trisaccharides.Cryoprotectants may also have lyoprotectant effects.

The term “agent suitable for keeping the pH in the range of 3 to 9”, or“buffering agent”, encompasses those agents that maintain the solutionpH in an acceptable range between 3.0 and 9.0. Typical examples ofagents capable of keeping the pH within a range of 3 to 9 are the acidform or salts of citric acid, acetic acid, histidine, malic acid,phosphoric acid, tartaric acid, succinic acid, MES, HEPES, PIPES,imidazole, TRIS, lactic acid, glutaric acid and glycylglycine. It is tobe understood that a combination of agents, wherein the combination ofagents is suitable for maintaining the pH in the above-described range,may also be used in the present invention.

The term “lyophilised cake” as used herein is denoted to encompass thesolid composition obtained upon processing a dissolved or at least apartly dissolved composition under conditions involving at least onestep of cooling said dissolved/partly dissolved composition to icefollowed by at least one step of vacuum drying.

The term “lyophilization” and “freeze-drying” encompasses a processduring which liquid is removed from a dissolved or at least partlydissolved composition under conditions involving at least one step ofcooling the dissolved or partly dissolved solution to ice followed byvacuum drying. Lyophilization, or freeze-drying, is the most commonprocess for making solid polypeptide pharmaceuticals. The processconsists of two major steps: freezing of a polypeptide solution, anddrying of the frozen solid under vacuum. The drying step is furtherdivided into two phases: primary and secondary drying. The primarydrying removes the frozen water (sublimation of ice) and the secondarydrying removes the non-frozen “bound” water (desorption of water). Moredetailed analysis of each lyophilization step is provided in, e.g., Wanget al, International Journal of Pharmaceutics 203 (2000): 1-60 (seesection 4, page 16 ff.).

Typically, a composition is freeze-dried by filling into vials, freezingon the shelves of the freeze-dryer, after which a vacuum is establishedand the shelves heated to implement primary drying (or sublimation ofice). Thereafter, secondary drying (or desorption of sorbed water) takesplace at a higher temperature until the completion of the process, i.e.,where the composition contains a sufficiently low content of moisture(water). Methods for freeze-drying are generally known in the art, see,for example, Wang et al, International Journal of Pharmaceutics 203(2000): 1-60.

It is within the ordinary skill of the practitioner to optimize thefreeze-drying conditions in regard of temperature(s), time(s) at eachtemperature, and also pressure that is to be used during the process fora specific composition.

The term “moisture content” is meant to encompass water associated withthe product, including, without limitation, water in adsorbed form, suchas unfrozen water entrapped in or adsorbed to the frozen solute phaseand/or associated with the amorphous phase or adsorbed to thecrystalline solid. The term “water content” is used interchangeably with“moisture content”. The desired residual moisture level (moisturecontent) is a function of the duration and the temperature of thesecondary drying step. Several methods for determining the residualmoisture content during lyophilization are known in the art; forexample, an electronic hygrometer or a residual gas analyser may beused. Moisture contents of freeze-dried formulations can be determinedby several methods known in the art, such as, for example,loss-on-drying, Karl Fischer titration, thermal gravimetric analysis(TGA), gas chromatography (GC), or near IR (see, e.g. Wang et al,International Journal of Pharmaceutics 203 (2000): 1-60). Methods fordetermining water contents (moisture contents) are also described inboth the European and U.S. Pharmacopoeias. For example, determination ofwater content can be performed by Karl Fischer coulometric titration asdescribed in the U.S. Pharmacopoeia (USP <921, Ic>) or the EuropeanPhamacopoeia (EP <2.5.32>).

In brief, the method is as follows:

Determination of Water Content by Coulometric Titration:

The Karl Fischer reaction is used in the coulometric determination ofwater based upon the quantitative reaction of water with sulphur dioxideand iodine in an anhydrous medium. Iodine is produced electrochemicallyin the reaction cell by oxidation of iodide. The iodine produced at theanode reacts immediately with the water and the sulphur dioxidecontained in the reaction cell. The amount of water in the substance isdirectly proportional to the quantity of electricity up until thetitration end-point. When all of the water in the cell has beenconsumed, the end-point is reached and thus an excess of iodine appearswhich is detected electrometrically thus indicating the end-point. Thepercentage water content present in the substance is then calculated.

Moisture content may be defined in terms of the weight of the sample inthe vial at the time of analysis (i.e. solids plus the waterpresent—called wet weight basis) or it may be defined in terms where itis corrected for the measured water in the sample (i.e. dry weightbasis). In case of freeze-dried products with low moisture contents thetwo measurements (wet weight basis vs. dry weight basis) yield verysimilar results. As used herein, moisture contents are defined in termsof the solids plus the water present (i.e., wet weight basis).

The term “bulking agent” generally includes agents, which provide goodlyophilised cake properties, which form a pharmaceutically elegantproduct, which help the polypeptide overcome various stresses,shear/freezing for example, associated with lyophilisation processes,and which help to maintain polypeptide activity levels during thefreeze-drying process and subsequent storage. Non-limiting examples ofbulking agents include mannitol, glycine, sucrose, lactose. These agentsmay also contribute to the tonicity of the formulations.

Isotonic solutions have a tonicity within the physiological range of theblood, peritoneal fluid or other relevant body fluids. By isotonicity ismeant a solution with an osmotic pressure corresponding to the osmoticpressure of a 0.9% NaCl solution (=286 mOsM). The term “essentiallyisotonic” is denoted to mean a tonicity corresponding to the osmoticpressure of a saline solution containing from about 0.7 to about 1.5%NaCl, such as, e.g., from about 0.8 to about 1.3%, about 0.8 to about1.1%, about 0.8 to about 1.0%, or about 0.9% NaCl.

The term “tonicity modifier” or “tonicity modifying agent” is denoted tomean any agent, or mixture of agents, capable of adjusting the tonicityof the composition such that upon dissolving the composition at the timeof use, the dissolved (or reconstituted) composition is essentiallyisotonic. Obviously, the tonicity of the reconstituted solution maydepend on both the contents of tonicity-modifying agents in the drycomposition and in the reconstitution solution.

Tonicity modifying agents include, without limitation, componentsselected from the list of: sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, mannitol, glycerol,propylene glycol, or mixtures of two or more of these.

Amounts of the above tonicity modifying agents suitable for providing acomposition having a tonicity as defined above are, for example, from 0to about 9 mg/ml of sodium chloride, from 0 to about 17 mg/ml of calciumchloride dihydrate, from 0 to about 51 mg/ml of mannitol, from 0 toabout 26 mg/ml of glycerol, from 0 to about 21 mg/ml of propyleneglycol, depending on whether the individual tonicity modifier is usedalone or in combination with one or more tonicity modifiers.

The term “surfactants” generally include those agents, which protect thepolypeptide from air/solution interface-induced stresses andsolution/surface induced-stresses. For example surfactants may protectthe polypeptide from aggregation. Suitable surfactants may include e.g.polysorbates, polyoxyethylene alkyl ethers such as Brij 35®, orpoloxamer such as Tween 20, Tween 80, or poloxamer 188. Preferredsurfactants are poloxamers, e.g. Poloxamer 188, Poloxamer 407;polyoxyethylene alkyl ethers, e.g. Brij 35®, Cremophor A25, SympatensALM/230; and polysorbates/Tweens, e.g. Polysorbate 20, Polysorbate 80.More preferred are Poloxamers, e.g. Poloxamer 188, and Tweens, e.g.Tween 20 and Tween 80. Typically, the surfactants are added in an amountof from 0.005 to 5 mg/ml. Preferred amounts are from 0.01 to 3 mg/ml,more preferred from 0.01 to 0.3 mg/ml for Tween 20 and/or Tween 80 andfrom 0.05 to 3.0 mg/ml for Poloxamer 188.

The term “initial content” relates to the amount of Factor VIIpolypeptides added to a composition at the time of preparation. Theconcentration given herein (mg/ml) refer to either the concentration inthe solution of Factor VII polypeptide before removing the moisture(e.g. before freeze-drying) or in the reconstituted composition, or isreferred as % w/w, which then relates to the concentration in the solidcomposition, e.g. the lyophilised cake.

As used herein, amounts specified are understood to be ± about 10%; thusabout 50 mM includes 50 mM±5 mM, 4% includes 4%±0.4%, etc.

As stated above, the present investigators have contributed essentiallyto the art by stabilising Factor VII polypeptides thereby allowinglong-term storage without causing increased risk and inconvenience tothe user.

The present investigators have found that a number of crucial parametersneed to be adjusted in stabilising Factor VII polypeptides. Oneimportant parameter relates, at least in part, to the moisture content,e.g. water. The moisture content should be limited. As a furtheressential parameter, the composition should at least include onestabilizing agent.

Stabilizing agents include, without limitation, antioxidants,saccharides, polyols, surfactants, and agents suitable for maintainingpH in a predetermined range.

In one embodiment of the present invention, a proper stabilizing agentincludes the combination of at least two groups of pharmaceuticallyacceptable excipients selected from the group consisting ofantioxidants, saccharides and polyols. The saccharides and polyols havelyoprotectant and/or cryoprotectant properties that may be important, atleast in part, in the event where the composition is freeze-dried. Ingeneral, improved stability may be achieved, in part, by the propercombination of at least two of these groups of excipients. However, morespecifically it was found that when said combination comprises asaccharide (sucrose) or an antioxidant (methionine), the stabilisingeffect may be even more significant. Moreover, it was also surprisinglyfound that methionine prevents oxidative degradation of the Factor VIIpolypeptides.

As stated, in one embodiment of the invention the stabilising agentincludes combining at least two groups of pharmaceutically acceptableexcipients.

According to the invention, the Factor VII polypeptide is meant toencompass the polypeptides as described above. In suitable embodimentsof the invention, the Factor VII polypeptide is selected from the groupconsisting of Human Factor VIIa, Recombinant Human Factor VIIa and aFactor VII Sequence Variant. Preferably, the Factor VII Polypeptide isHuman Factor VIIa or Recombinant Human Factor VIIa or a FactorVII-related polypeptide wherein the ratio between the activity of saidFactor VII-related polypeptide and wild-type Factor VII is at least 1.25when tested in one or more of the “In Vitro Proteolysis Assay” and the“in Vitro Hydrolysis Assay” as described in the present specification.

As stated, the moisture content should be limited. For the purposes ofthe present invention, the Factor VII polypeptides, when provided inbulk, may be provided in solid or liquid form. However, typically theFactor VII polypeptides, when provided in bulk, are in liquid form.Thus, further processing of the bulk polypeptides for the manufacturingof compositions requires the steps of adding suitable excipients andremoving the liquid from the bulk, said addition of excipients may becarried out before or after removing the liquid. One such mean forremoving liquid from a polypeptide relates to freeze-drying. Therefore,in a preferred embodiment of the present invention, the composition isin the form of a lyophilised cake. However, the present invention doesnot preclude other processes that are suitable for removing the liquidfrom the bulk polypeptide so as to achieve a solid composition withmoisture content of not more than about 3% w/w.

Moreover, according to the invention, the moisture content is preferablynot more than about 2.5% w/w, preferably not more than about 2% w/w,most preferably not more than about 1.5% w/w.

As may be understood, the invention relates, in part, to limiting thedegradation of Factor VII polypeptides during preparation, e.g. duringadmixing of excipients and removing of liquid so as to achieve a solidcomposition with moisture content of the most 3% w/w, and to limitingsaid degradation from the time of manufacturing the solid compositionuntil the time of use, e.g. until the time when the composition is to beadministered by a patient.

Therefore, as a still further parameter in stabilising kits andcompositions comprising Factor VII polypeptides, the pH should be keptin the pH range within 3 to 9 when dissolved in aqueous solvent, suchas, e.g., pure water or aqueous buffer. That is to say that the pH inthe polypeptide solution at the time before removing the moisturecontent, e.g. before freeze-drying, should be kept within a pH of about3 to about 9. Advantageously, this pH range is also within the desiredphysiological range, thereby causing no harm to the user uponadministering the composition by parenteral means. Preferably, the pH ofthe solution is from about 4.0 to about 9.0, such as 4.0 to 8.0, 4.0 to7.5, 4.0 to 7.0, 4.5 to 7.0, 4.5 to 6.8, 4.5 to 6.5, 5.0 to 7.0, 5.0 to6.5, 5.0 to 6.0, 5.5 to 6.0, or about 5.5 to about 6.5 such as about5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

In suitable embodiments of the invention, the agent suitable for keepingthe pH in the range of 3 to 9 is selected from the group consisting ofacid or salts of citric acid, acetic acid, histidine, malic acid,phosphoric acid, tartaric acid, succinic acid, MES, HEPES, imidazole,TRIS, lactic acid, glutaric acid, PIPES and glycylglycine, or a mixtureof at least two such listed agents, wherein the mixture is able toprovide a pH value in the specified range.

Furthermore, the suitable agent for keeping the pH in the range of 3 to9 may also be a mixture of at least two such listed agents, wherein themixture is able to provide a pH value in the specified range. Theconcentration of the suitable agents is in the range of from about 0.1mM to 100 mM; from about 0.1 mM to about 50 mM; such as from about 0.1mM to about 40 mM; from about 0.1 mM to about 35 mM; from about 0.1 mMto about 30 mM; from about 0.5 mM to about 25 mM; from about 1 mM toabout 20 mM; from about 1 mM to about 15 mM; from about 5 mM to about 20mM; or from about 5 mM to about 15 mM.

In one embodiment of the invention, the agent suitable for keeping thepH in the range of 3 to 9 is histidine, preferably L-histidine.

Degradation of the Factor VII polypeptide by the oxidative pathway aswell as by the aggregation pathway are sensitive parameters ofstability.

Typically, the compositions are stabilised upon termination of thefreeze-drying such that less than 5% w/w, such as less than 4, 3 or 2%w/w of the initial content of Factor VII polypeptide is converted intoits oxidised forms. The initial content of said Factor VII polypeptidebeing the amount added to the composition upon preparation of thecomposition before the freeze-drying step. Moreover, less than 5% w/w,such as less than 4.0%, 3.0%, 2.5%, 2%, 1.5%, or less than 1% w/w of theinitial content of Factor VII polypeptide is recovered as aggregateforms, as determined by conventional analytical methods (such as, forexample, as described in the Examples of the present application).

The present investigators have found that further degradation (i.e., ascalculated from the time of termination of the manufacturing processuntil 8 months of storage at 30° C.) of a Factor VII polypeptide isminimal upon storage under ambient conditions. It was found thatcompositions comprising an antioxidant (methionine) are more stabletowards oxidative degradation of the Factor VII polypeptide.

Therefore, suitable compositions have a limited increase in the contentof oxidised forms upon storage for at least 8 months at ambientconditions.

That is to say that in still more interesting embodiments, thecomposition is stable such that no more than about 6% w/w of the initialcontent of Factor VII polypeptide is additionally degraded into oxidisedforms upon storage of the composition for 8 months at 30° C. aftertermination of the manufacturing process, e.g. freeze-drying process. Infurther suitable embodiments thereof, not more than about 5, 4, 3, 2, or1.5% w/w or of the Factor VII polypeptide is additionally converted intooxidised forms, as calculated from the time of termination of themanufacturing process until 8 months of storage at 30° C. In theseembodiments of the invention the compositions are stable such that notmore than about 5% (4, 3, 2, or 1.5%) w/w of the initial content ofFactor VII polypeptide is converted to oxidised forms upon storage ofsaid composition at 30° C. for 8 months. As stated above, the initialcontent relates to the amount of Factor VII polypeptide added to thecomposition upon preparation of the composition before the freeze-dryingstep.

As indicated, the degradation of Factor VII polypeptides by theaggregation pathway may also be regarded as an essential stabilityindicating parameter.

Thus, interesting embodiments of the invention relate to compositionsthat are stable such that not more than about 5% w/w of the initialcontent of Factor VII polypeptide is converted to aggregates uponstorage of said composition at 30° C. for 8 months. As stated above theinitial content of said Factor VII polypeptide being the amount added tothe composition upon preparation of the composition before thefreeze-drying step. By proper optimisation of, at least in part, thecontents of saccharides, polyols and antioxidants, the composition isstable such that not more than about 4.0%, 3.0% w/w, such as 2.5, 2.0,1.5, or 1.0% w/w, of the initial content of Factor VII polypeptide isconverted to aggregates upon storage of said composition at 30° C. for 8months.

Thus, advantageously, the compositions of the invention have lowcontents of oxidised forms and aggregates upon termination of themanufacturing process, i.e. upon termination of the freeze-dryingprocess, and thus the compositions according to the invention arecharacterised by having a low initial content of oxidised forms andaggregates before being subjected to storage, e.g. not more than about5% w/w, such as 4%, 3%, or 2% w/w of the initial contents of Factor VIIpolypeptide is converted into an oxidised form, and less than 5% w/w,such as not more than about 4.0%, 3.0%, 2.5%, 2%, 1.5%, or not more thanabout 1% w/w, is converted into a dimeric or higher-order polymeric formupon termination of the manufacturing process

Moreover and advantageously, the kits and compositions of the inventionare storage-stable, e.g. less than 10% w/w, such as 6%, 5%, 4%, 3%, 2%,or 1.5% w/w of the initial contents of Factor VII polypeptide isconverted into an oxidised form, and less than 5% w/w, such as 4%, 3%,2.5%, 2%, 1.5%, or 1% w/w is converted into a dimeric or higher-orderpolymeric form upon storage at 30° C. for at least 8 months in the dark.

As mentioned, said improved stability relates to the proper combinationof particular excipients. According to the present invention, thestabilising agents may be selected from the group of saccharides,polyols and antioxidants. In suitable embodiments, the saccharides ofinterest are di- and tri-saccharides and polysaccharides such that thesaccharides may be selected from the group consisting of sucrose,dextrose, lactose, maltose, trehalose, cyclodextrins, maltodextrins anddextrans. Moreover, in some embodiments, the polyol is selected from thegroup consisting of mannitol, sorbitol and xylitol. In still interestingembodiments, the antioxidant is selected from the group consisting ofhomocysteine, cysteine, cystathionine, methionine, gluthatione, andpeptides containing any one of homocysteine, cysteine, cystathionine,methionine and gluthatione.

It is understood that the saccharide and polyol excipients,respectively, may also be a mixture of at least two such listed agents.In one series of embodiments of the invention, the saccharide excipientused is a combination of at least two di-, tri- and/or polysaccharides,such as, for example, sucrose in combination with cyclodextrin,trehalose in combination with cyclodextrin, sucrose in combination withdextran, or sucrose in combination with lactose. In one series ofembodiments of the invention, the polyol excipient used is a combinationof at least two polyols, such as, for example, mannitol in combinationwith sorbitol, mannitol in combination with xylitol, or sorbitol incombination with xylitol. In one series of embodiments of the invention,the antioxidant excipient used is a combination of at least twoantioxidants, such as, for example, methionine in combination with oneor more of homocysteine, cysteine, cystathionine, gluthatione, andpeptides containing any one of homocysteine, cysteine, cystathionine,methionine and gluthatione.

In particular interesting embodiments, the antioxidant is selected fromthe group consisting of homocysteine, cysteine, cystathionine,methionine, gluthatione, and peptides containing any one ofhomocysteine, cysteine, cystathionine, methionine and gluthatione. In apreferred embodiment, the antioxidant is methionine.

In further interesting embodiments of the invention, the polyols are tobe present in an amount ranging from about 5% w/w to about 90% w/w.Preferably, the amount of the polyol is to be present in a range fromabout 18% w/w to about 88% w/w, such as from about 18% w/w to about 83%w/w, 25% to 80%, 30% to 65%, 30% to 80%, 40% to 80%, 50% to 80%, 30% to75%, 40% to 75%, 50% to 75%, or from about 50% to about 70% w/w.

The polyol are to be present in an amount ranging from about 0.5 to 75mg/ml, such as from about 2 to 60 mg/ml, 5 mg/ml to 55 mg/ml, 8 to 45mg/ml, 10 to 40 mg/ml, 10 to 30 mg/ml, or from about 2 to 45 mg/ml, 5mg/ml to 45 mg/ml, 5 to 35 mg/ml, 5 to 25 mg/ml, 5 to 20 mg/ml, 20 to 40mg/ml, or such as from about 20 to 30 mg/ml,

Moreover, in interesting embodiments thereof as well as in some otherinteresting embodiments of the invention, the saccharide is to bepresent in the composition in an amount ranging from about 0 to about85% w/w. In further interesting embodiments thereof, the amount rangesfrom about 3% w/w to about 80% w/w, such as from about 7% w/w to about75% w/w, 10% to 70%, 10% to 50%, 20% to 50%, 10% to 40%, or from about10% w/w to about 35% w/w.

The saccharide should be in an amount ranging from about 0.5 to 75mg/ml, such as from about 2 to 60 mg/ml, from about 5 mg/ml to 55 mg/ml,from about 8 to 45 mg/ml, from about 10 to 40 mg/ml, from about 10 to 30mg/ml, or from about 2 to 45 mg/ml, from about 5 mg/ml to 45 mg/ml, fromabout 5 to 35 mg/ml, from about 5 to 25 mg/ml, such as from about 5 to20 mg/ml.

The antioxidant should be provided in an amount ranging from about 0.05to 10 mg/ml, preferably from about 0.1 to 5 mg/ml, more preferably fromabout 0.1 mg/ml to 2.5 mg/ml, even more preferably from about 0.1 to 2mg/ml, most preferably from about 0.1 to 1 mg/ml.

The ratio between the polyol and the saccharide needs to be properlyadjusted. In suitable embodiments of the invention, said polyol is in aweight ratio relative to said saccharide ranging from about 100:1 to1:50. In even more suitable embodiments thereof, said weight ratio isfrom about 50:1 to 1:10, more preferably from about 20:1 to 1:5. Inother suitable embodiments, the weight ratio relates to ranges fromabout 10:1 to 1:2, and from about 6:1 to 1:2. Suitable embodimentsrelate to those wherein said sugar alcohol is in a weight ratio relativeto said saccharide ranging from about 4:1 to 1:1, such as from about 4:1to 3:2 or from about 1:1 to 3:2.

In some embodiments of the invention, the polyol is mannitol and instill further embodiments the saccharide is sucrose. Moreover, in stillfurther embodiments the antioxidant is methionine.

In still preferred embodiments of the invention, the composition furthercomprises other pharmaceutical excipients acting as bulking agent. Thatis to say that bulking agents other than mannitol are included in thecompositions. In particular, bulking agents are included in compositionsprepared by freeze-drying.

Initial contents of Factor VII polypeptide in the composition ispreferably from about 0.6 mg/mL to about 10.0 mg/mL, such as from about0.6 mg/mL to about 8 mg/mL, from about 0.6 mg/mL to about 6 mg/mL, fromabout 0.6 mg/mL to about 5 mg/mL, from about 0.6 mg/mL to about 4 mg/mL,from about 0.6 mg/mL to about 3 mg/mL, from about 1.0 mg/mL to about 5mg/mL, from about 1.0 mg/mL to about 4 mg/mL, or from about 1.0 mg/mL toabout 3 mg/mL, e.g., about 1.0 mg/mL, about 2.0 mg/mL, about 3.0 mg/mL,about 4.0 mg/mL, or about 5.0 mg/mL.

In one embodiment, the composition contained in the first unit form ofthe kit comprises: Factor VII polypeptide, Mannitol, Sucrose, andpolysorbate, preferably polysorbate 20 or 80, has a moisture content ofnot more than about 3%, and has a pH in the range of 5.0 to 7.0 when thecomposition is dissolved in water. In one embodiment, the compositionfurther comprises one or more components selected from the list of:CaCl2, NaCl, and Glycylglycine. In one embodiment, the Factor VIIpolypeptide is human Factor VIIa.

In one embodiment, the composition contained in the first unit form ofthe kit comprises: Factor VII polypeptide, Mannitol, Sucrose,methionine, and polysorbate, preferably polysorbate 20 or 80, has amoisture content of not more than about 3%, and has a pH in the range of5.0 to 7.0 when the composition is dissolved in water. In oneembodiment, the composition further comprises one or more componentsselected from the list of: CaCl2, NaCl, and Glycylglycine. In oneembodiment, the Factor VII polypeptide is human Factor VIIa.

In another embodiment, the composition contained in the first unit formof the kit comprises Factor VII polypeptide, Mannitol, Sucrose,Histidine, and polysorbate, preferably polysorbate 20 or 80, has amoisture content of not more than about 3%, and has a pH in the range of5.0 to 7.0 when the composition is dissolved in water. In oneembodiment, the composition further comprises one or more componentsselected from the list of: CaCl2, NaCl, and Glycylglycine. In oneembodiment, the Factor VII polypeptide is human Factor VIIa.

In one embodiment, the composition contained in the first unit form ofthe kit comprises: Factor VII polypeptide, Mannitol, Sucrose,methionine, Histidine, and polysorbate, preferably polysorbate 20 or 80,has a moisture content of not more than about 3%, and has a pH in therange of 5.0 to 7.0 when the composition is dissolved in water. In oneembodiment, the composition further comprises one or more componentsselected from the list of: CaCl2, NaCl, and Glycylglycine. In oneembodiment, the Factor VII polypeptide is human Factor VIIa.

In another embodiment, the composition contained in the first unit formof the kit comprises Factor VII polypeptide, Mannitol, Sucrose, andPoloxamer 188, has a moisture content of not more than about 3%, and hasa pH in the range of 5.0 to 7.0 when the composition is dissolved inwater. In one embodiment, the composition further comprises one or morecomponents selected from the list of: CaCl2, NaCl, and Glycylglycine. Inone embodiment, the Factor VII polypeptide is human Factor VIIa.

In another embodiment, the composition contained in the first unit formof the kit comprises Factor VII polypeptide, Mannitol, Sucrose,methionine, and Poloxamer 188, has a moisture content of not more thanabout 3%, and has a pH in the range of 5.0 to 7.0 when the compositionis dissolved in water. In one embodiment, the composition furthercomprises one or more components selected from the list of: CaCl2, NaCl,and Glycylglycine. In one embodiment, the Factor VII polypeptide ishuman Factor VIIa.

In another embodiment, the composition contained in the first unit formof the kit comprises Factor VII polypeptide, Mannitol, Sucrose,Histidine, and Poloxamer 188, has a moisture content of not more thanabout 3%, and has a pH in the range of 5.0 to 7.0 when the compositionis dissolved in water. In one embodiment, the composition furthercomprises one or more components selected from thelist of: CaCl2, NaCl,and Glycylglycine. In one embodiment, the Factor VII polypeptide ishuman Factor VIIa.

In another embodiment, the composition contained in the first unit formof the kit comprises Factor VII polypeptide, Mannitol, Sucrose,Histidine, methionine, and Poloxamer 188, has a moisture content of notmore than about 3%, and has a pH in the range of 5.0 to 7.0 when thecomposition is dissolved in water. In one embodiment, the compositionfurther comprises one or more components selected from the list of:CaCl2, NaCl, and Glycylglycine. In one embodiment, the Factor VIIpolypeptide is human Factor VIIa.

In one embodiment, the administration vehicle contained in the secondunit form of the kit comprises: Water, and histidine. In a furtherembodiment, the vehicle further comprises one or more componentsselected from the list of: CaCl2, NaCl, and Glycylglycine. In a furtherembodiment thereof, the vehicle comprises one or more componentsselected from the list of: CaCl2 in a concentration of about 5-15 mM,NaCl in a concentration of about 30 to 60 mM, such as, e.g., about 40 mMor about 50 mM.

In a preferred embodiment, the method for preparing a stable Factor VIIpolypeptide comprises freeze-drying. The freeze-drying relates to aprocess, wherein the solution comprising said Factor VII polypeptide isfilled into lyophilisation vials or the like. Said Factor VIIpolypeptide may optionally be subjected to sterile filtration beforestart of freeze-drying. Cooling is applied to the shelves of thefreeze-drier in order to freeze the vials and the solution belowcritical product temperatures. Water is removed by introducing vacuumand condensation of water vapour on the ice-condenser of thefreeze-drier. When the product is dry, usually less than 3% residualmoisture content (e.g., measured by Karl Fischer coulometric titrationas described above), the vials are closed and capped. Manufacturing isfinalised and the composition is now in a form of a lyophilised cake.

The reconstituted compositions are intended for parenteraladministration for prophylactic and/or therapeutic treatment.Preferably, the pharmaceutical compositions are administeredparenterally, i.e., intravenously, subcutanously, or intramuscularly, orthey are administered by way of continuous or pulsative infusion.

Therefore, a still further aspect of the invention relates to the use ofthe solid stabilised composition for the preparation of a medicament fortreating a coagulation factor-responsive syndrome. In one embodiment,the invention relates to the use of Factor VII polypeptide for thepreparation of a medicament for treating a Factor VII-responsivesyndrome.

In different embodiments, said Factor VII-responsive syndrome ishaemophilia A, haemophilia B, Factor XI deficiency, Factor VIIdeficiency, thrombocytopenia, von Willebrand's disease, presence of aclotting Factor inhibitor, surgery or trauma. Additionally, FactorVII-responsive syndrome may be associated with anticoagulant therapy.

As stated the compositions of the invention is in solid form.Accordingly, in a suitable embodiment the medicament should be suitablefor being dissolved, which allows for parenteral administration of themedicament. Thus, when administering the compositions to a patient, itcomprises the step of dissolving the composition in a suitable liquidprior to the administering step.

Abbreviations Used Herein:

-   FVII: Coagulation Factor VII in its single chain form-   FVIIa: Coagulation Factor VII in its cleaved, activated two-chain    form-   rFVII (rFVIIa): Recombinant Factor VII (recombinant Factor VIIa)

Embodiments

In one series of embodiments of the invention, the first unit form ofthe kit comprises the excipients, and amounts thereof, and has the pH asshown in the list of formulations 1 to 48:

TABLE 1 Composition 1 2 3 4 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0mg/mL Sodium chloride 2.92 mg/mL 2.92 mg/mL 2.92 mg/mL 2.92 mg/mLCalcium chloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL 1.47 mg/mLGlycylglycine 1.32 mg/mL 1.32 mg/mL — — L-Histidine — 1.55 mg/mL — 1.55mg/mL Polysorbate 80 0.07 mg/mL 0.07 mg/mL 0.07 mg/mL 0.07 mg/mLMethionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH5.50 5.50 5.50 5.50 Composition 5 6 7 8 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0mg/mL 1.0 mg/mL Sodium chloride — — — — Calcium chloride 2H20 1.47 mg/mL1.47 mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine 1.32 mg/mL 1.32 mg/mL — —L-Histidine — 1.55 mg/mL — 1.55 mg/mL Polysorbate 80 0.07 mg/mL 0.07mg/mL 0.07 mg/mL 0.07 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10mg/mL 10 mg/mL 10 mg/mL pH 5.50 5.50 5.50 5.50 Composition 9 10 11 12rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Sodium chloride 2.34mg/mL 2.34 mg/mL 2.34 mg/mL 2.34 mg/mL Calcium chloride 2H20 1.47 mg/mL1.47 mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine 1.32 mg/mL 1.32 mg/mL — —L-Histidine — 1.55 mg/mL — 1.55 mg/mL Polysorbate 80 0.07 mg/mL 0.07mg/mL 0.07 mg/mL 0.07 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10mg/mL 10 mg/mL 10 mg/mL pH 5.50 5.50 5.50 5.50 Composition 13 14 15 16rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Sodium chloride 2.92mg/mL 2.92 mg/mL 2.92 mg/mL 2.92 mg/mL Calcium chloride 2H20 1.47 mg/mL1.47 mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine 1.32 mg/mL 1.32 mg/mL — —L-Histidine — 1.55 mg/mL — 1.55 mg/mL Polysorbate 80 0.07 mg/mL 0.07mg/mL 0.07 mg/mL 0.07 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10mg/mL 10 mg/mL 10 mg/mL pH 6.0 6.0 6.0 6.0 Composition 17 18 19 20rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Sodium chloride — — — —Calcium chloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL 1.47 mg/mLGlycylglycine 1.32 mg/mL 1.32 mg/mL — — L-Histidine — 1.55 mg/mL — 1.55mg/mL Polysorbate 80 0.07 mg/mL 0.07 mg/mL 0.07 mg/mL 0.07 mg/mLMethionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH6.0 6.0 6.0 6.0 Composition 21 22 23 24 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0mg/mL 1.0 mg/mL Sodium chloride 2.34 mg/mL 2.34 mg/mL 2.34 mg/mL 2.34mg/mL Calcium chloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL 1.47 mg/mLGlycylglycine 1.32 mg/mL 1.32 mg/mL — — L-Histidine — 1.55 mg/mL — 1.55mg/mL Polysorbate 80 0.07 mg/mL 0.07 mg/mL 0.07 mg/mL 0.07 mg/mLMethionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH6.0 6.0 6.0 6.0 Composition 25 26 27 28 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0mg/mL 1.0 mg/mL Sodium chloride 2.92 mg/mL 2.92 mg/mL 2.92 mg/mL 2.92mg/mL Calcium chloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL 1.47 mg/mLGlycylglycine 1.32 mg/mL 1.32 mg/mL — — L-Histidine — 1.55 mg/mL — 1.55mg/mL Polysorbate 80 0.1 mg/mL 0.1 mg/mL 0.1 mg/mL 0.1 mg/mL Methionine0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25 mg/mL 25mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH 5.50 5.505.50 5.50 Composition 29 30 31 32 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL1.0 mg/mL Sodium chloride — — — — Calcium chloride 2H20 1.47 mg/mL 1.47mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine 1.32 mg/mL 1.32 mg/mL — —L-Histidine — 1.55 mg/mL — 1.55 mg/mL Polysorbate 80 0.1 mg/mL 0.1 mg/mL0.1 mg/mL 0.1 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mLMannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL10 mg/mL 10 mg/mL pH 5.50 5.50 5.50 5.50 Composition 33 34 35 36 rFVIIa1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Sodium chloride 2.34 mg/mL 2.34mg/mL 2.34 mg/mL 2.34 mg/mL Calcium chloride 2H20 1.47 mg/mL 1.47 mg/mL1.47 mg/mL 1.47 mg/mL Glycylglycine 1.32 mg/mL 1.32 mg/mL — —L-Histidine — 1.55 mg/mL — 1.55 mg/mL Polysorbate 80 0.1 mg/mL 0.1 mg/mL0.1 mg/mL 0.1 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mLMannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL10 mg/mL 10 mg/mL pH 5.50 5.50 5.50 5.50 Composition 37 38 39 40 rFVIIa1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Sodium chloride 2.92 mg/mL 2.92mg/mL 2.92 mg/mL 2.92 mg/mL Calcium chloride 2H20 1.47 mg/mL 1.47 mg/mL1.47 mg/mL 1.47 mg/mL Glycylglycine 1.32 mg/mL 1.32 mg/mL — —L-Histidine — 1.55 mg/mL — 1.55 mg/mL Polysorbate 80 0.1 mg/mL 0.1 mg/mL0.1 mg/mL 0.1 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mLMannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL10 mg/mL 10 mg/mL pH 6.0 6.0 6.0 6.0 Composition 41 42 43 44 rFVIIa 1.0mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Sodium chloride — — — — Calciumchloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine1.32 mg/mL 1.32 mg/mL — — L-Histidine — 1.55 mg/mL — 1.55 mg/mLPolysorbate 80 0.1 mg/mL 0.1 mg/mL 0.1 mg/mL 0.1 mg/mL Methionine 0.5mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH 6.0 6.0 6.0 6.0Composition 45 46 47 48 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mLSodium chloride 2.34 mg/mL 2.34 mg/mL 2.34 mg/mL 2.34 mg/mL Calciumchloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine1.32 mg/mL 1.32 mg/mL — — L-Histidine — 1.55 mg/mL — 1.55 mg/mLPolysorbate 80 0.1 mg/mL 0.1 mg/mL 0.1 mg/mL 0.1 mg/mL Methionine 0.5mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH 6.0 6.0 6.0 6.0

In another series of embodiments, the first unit form of the kitcomprises the excipients, and amounts thereof, and has the pH as shownin the list of formulations 100 to 124:

TABLE 2 Composition 100 102 103 104 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL1.0 mg/mL Sodium chloride 2.92 mg/mL 2.92 mg/mL 2.92 mg/mL 2.92 mg/mLCalcium chloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL 1.47 mg/mLGlycylglycine 1.32 mg/mL 1.32 mg/mL — — L-Histidine — 1.55 mg/mL — 1.55mg/mL Poloxamer 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Methionine 0.5mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH 5.50 5.50 5.505.50 Composition 105 106 107 108 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL1.0 mg/mL Sodium chloride — — — — Calcium chloride 2H20 1.47 mg/mL 1.47mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine 1.32 mg/mL 1.32 mg/mL — —L-Histidine — 1.55 mg/mL — 1.55 mg/mL Poloxamer 1.0 mg/mL 1.0 mg/mL 1.0mg/mL 1.0 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mLMannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL10 mg/mL 10 mg/mL pH 5.50 5.50 5.50 5.50 Composition 109 110 111 112rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Sodium chloride 2.34mg/mL 2.34 mg/mL 2.34 mg/mL 2.34 mg/mL Calcium chloride 2H20 1.47 mg/mL1.47 mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine 1.32 mg/mL 1.32 mg/mL — —L-Histidine — 1.55 mg/mL — 1.55 mg/mL Poloxamer 1.0 mg/mL 1.0 mg/mL 1.0mg/mL 1.0 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mLMannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL10 mg/mL 10 mg/mL pH 5.50 5.50 5.50 5.50 Composition 113 114 115 116rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Sodium chloride 2.92mg/mL 2.92 mg/mL 2.92 mg/mL 2.92 mg/mL Calcium chloride 2H20 1.47 mg/mL1.47 mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine 1.32 mg/mL 1.32 mg/mL — —L-Histidine — 1.55 mg/mL — 1.55 mg/mL Poloxamer 1.0 mg/mL 1.0 mg/mL 1.0mg/mL 1.0 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mLMannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL10 mg/mL 10 mg/mL pH 6.0 6.0 6.0 6.0 Composition 117 118 119 120 rFVIIa1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Sodium chloride — — — — Calciumchloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine1.32 mg/mL 1.32 mg/mL — — L-Histidine — 1.55 mg/mL — 1.55 mg/mLPoloxamer 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Methionine 0.5 mg/mL0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL 25mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH 6.0 6.0 6.0 6.0Composition 121 122 123 124 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0mg/mL Sodium chloride 2.34 mg/mL 2.34 mg/mL 2.34 mg/mL 2.34 mg/mLCalcium chloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL 1.47 mg/mLGlycylglycine 1.32 mg/mL 1.32 mg/mL — — L-Histidine — 1.55 mg/mL — 1.55mg/mL Poloxamer 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Methionine 0.5mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH 6.0 6.0 6.0 6.0

In one series of embodiments, the concentration of FVII polypeptide inany one of compositions 1 to 48 and 100 to 124 is from about 0.6 mg/mLto about 10.0 mg/mL, such as from about 0.6 mg/mL to about 8 mg/mL, fromabout 0.6 mg/mL to about 5 mg/mL, from about 0.6 mg/mL to about 3 mg/mL,from about 1.0 mg/mL to about 5 mg/mL, or from about 1.0 mg/mL to about3 mg/mL.

In another series of embodiments, the concentration of FVII polypeptidein any one of compositions 1 to 48 and 100 to 124 is selected from thelist of: about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9mg/mL, about 1.0 mg/mL, about 1.1 mg/mL, about 1.2 mg/mL, about 1.3mg/mL, about 1.4 mg/mL, about 1.5 mg/mL, about 1.6 mg/mL, about 1.7mg/mL, about 1.8 mg/mL, about 1.9 mg/mL, about 2.0 mg/mL, about 2.1mg/mL, about 2.2 mg/mL, about 2.3 mg/mL, about 2.4 mg/mL, about 2.5mg/mL, about 2.6 mg/mL, about 2.7 mg/mL, about 2.8 mg/mL, about 2.9mg/mL, about 3.0 mg/mL, about 3.1 mg/mL, about 3.2 mg/mL, about 3.3mg/mL, about 3.4 mg/mL, about 3.5 mg/mL, about 3.6 mg/mL, about 3.7mg/mL, about 3.8 mg/mL, about 3.9 mg/mL, and about 4.0 mg/mL

In one series of embodiments, the concentration of polysorbate informulations 1 to 48 and 100 to 124 is from about 0.05 to 0.08 mg/mL,such as, from about 0.06 to 0.08 mg/mL, or about 0.07 mg/mL.

In a another series of embodiments, the first unit forms comprise acomposition as described in any one of compositions 1 to 48 and 100 to124, and the second unit form comprises L-histidine in an amount of fromabout 0.5 mg/mL to 3 mg/mL, such as, from about 1.0 to about 2.0 mg/mL,or about 1.55 mg/ML.

In yet another series of embodiments, the first unit forms comprise acomposition as described in any one of compositions 1 to 48 and 100 to124, and the second unit form comprises from about 30 to about 60 mMNaCl, such as, about 40 mM, about 45 mM, or about 50 mM NaCl.

In yet another series of embodiments, the first unit forms comprise acomposition as described in any one of compositions 1 to 48 and 100 to124, and the second unit form comprises from about 30 to about 60 mMNaCl, such as, about 40 mM, about 45 mM, or about 50 mM NaCl; andL-histidine in an amount of from about 0.5 mg/mL to 3 mg/mL, such as,from about 1.0 to about 2.0 mg/mL, or about 1.55 mg/ML.

Another aspect of the invention is the provision of novel compositionscomprising a Factor VII polypeptide and at least one stabilizing agentselected from the group consisting of

-   a) a combination of an antioxidant and mannitol;-   b) a combination of methionine and a polyol;-   c) a combination of a saccharide and mannitol;-   d) a combination of sucrose and a polyol;-   e) methionine; and-   a polysorbate surfactant in an amount of from about 0.06 to 0.08    mg/mL;-   said composition having a moisture content of not more than about    3%;

In one embodiment, the combination of an antioxidant and mannitol (a)further comprises a saccharide. In another embodiment, the combinationof methionine and a polyol (b) further comprises a saccharide. Inanother embodiment, the combination of a saccharide and mannitol (c)further comprises an antioxidant. In another embodiment, the combinationof sucrose and a polyol (d) further comprises an antioxidant.

In an interesting embodiment, the antioxidant is selected from the groupconsisting of homocysteine, cysteine, cystathionine, methionine,gluthatione, and peptides containing any one of homocysteine, cysteine,cystathionine, methionine and gluthatione, or mixtures thereof;preferably methionine, or mixtures containing methionine. In anotherinteresting embodiment, the the saccharide is selected from the groupconsisting of sucrose, dextrose, lactose, maltose, trehalose,cyclodextrins, maltodextrins and dextrans, or mixtures thereof;preferably sucrose, or mixtures containing sucrose. In yet anotherinteresting imbodiment, the polyol is selected from the group consistingof mannitol, sorbitol and xylitol, or mixtures thereof; preferablymannitol, or mixtures containing mannitol.

The compositions may further comprise an agent suitable for keeping thepH of said composition in the range of 3 to 9 when dissolved in aqueoussolvent. Non-limiting examples of such agents as well as preferred pHranges have been described above.

The composition may further comprise a tonicity modifier.Non-limitingexamples of such tonicity modifiers have been describedabove.

The polysorbate surfactant is selected from the group consisting ofpolysorbate 20 or 80, preferably polysorbate 80.

In one embodiment of the invention, the novel compositions are selectedfrom the list of:

TABLE 3 Compound Formulation I Formulation II FVII polypeptide 0.6 to 10mg/ml 0.6 to 10 mg/ml Mannitol 20 to 40 mg/ml 20 to 40 mg/ml Sucrose 5to 20 mg/ml — Methionine 0-1 mg/ml 0-1 mg/ml Polysorbate 0.01 to 0.09mg/ml 0.01 to 0.09 mg/ml pH 5.0 to 7.0 5.0 to 7.0

In one embodiment thereof, the compositions are selected from the listof:

TABLE 4 Compound Formulation III Formulation IV FVII polypeptide 0.6 to3.0 mg/ml 0.6 to 3.0 mg/ml Mannitol 20 to 40 mg/ml 20 to 40 mg/mlSucrose 5 to 20 mg/ml — Methionine 0-1 mg/ml 0-1 mg/ml Polysorbate 0.01to 0.09 mg/ml 0.01 to 0.09 mg/ml pH 5.0 to 7.0 5.0 to 7.0

In one embodiment thereof, the compositions are selected from the listof:

TABLE 5 Formulation Formulation Compound Formulation V VI VII FVII 0.6to 3.0 mg/ml 0.6 to 3.0 mg/ml 0.6 to 3.0 mg/ml polypeptide Mannitol 25mg/ml 25 mg/ml 25 mg/ml Sucrose 10 mg/ml — 10 mg/ml Methionine 0-1 mg/ml0-1 mg/ml 0.5 mg/ml Polysorbate 0.01 to 0.09 mg/ml 0.01 to 0.09 mg/ml0.01 to 0.09 mg/ml pH 5.5 to 6.5 5.5 to 6.5 5.5 to 6.5 FormulationFormulation Compound VIII IX Formulation X FVII 0.6 to 3.0 mg/ml 0.6 to3.0 mg/ml 0.6 to 3.0 mg/ml polypeptide Mannitol 25 mg/ml 25 mg/ml 25mg/ml Sucrose — 10 mg/ml — Methionine 0.5 mg/ml — — Polysorbate 0.01 to0.09 mg/ml 0.01 to 0.09 mg/ml 0.01 to 0.09 mg/ml pH 5.5 to 6.5 5.5 to6.5 5.5 to 6.5 Formulation Formulation Formulation Compound XI XII XIIIFVII 0.6 to 1.5 mg/ml 0.6 to 1.5 mg/ml 0.6 to 1.5 mg/ml polypeptideMannitol 25 mg/ml 25 mg/ml 25 mg/ml Sucrose 10 mg/ml — 10 mg/mlMethionine 0-1 mg/ml 0-1 mg/ml 0.5 mg/ml Polysorbate 0.01 to 0.09 mg/ml0.01 to 0.09 mg/ml 0.01 to 0.09 mg/ml pH 5.5 to 6.5 5.5 to 6.5 5.5 to6.5 Formulation Formulation Formulation Compound XIV XV XVI FVII 0.6 to1.5 mg/ml 0.6 to 1.5 mg/ml 0.6 to 1.5 mg/ml polypeptide Mannitol 25mg/ml 25 mg/ml 25 mg/ml Sucrose — 10 mg/ml — Methionine 0.5 mg/ml — —Polysorbate 0.01 to 0.09 mg/ml 0.01 to 0.09 mg/ml 0.01 to 0.09 mg/ml pH5.5 to 6.5 5.5 to 6.5 5.5 to 6.5

In one series of embodiments, the concentration of polysorbate informulations I to XVI is from about 0.05 to 0.08 mg/mL, such as, fromabout 0.06 to 0.08 mg/mL, or about 0.07 mg/mL.

In one series of embodiments, the polysorbate in formulations I to XVIis polysorbate 20, e.g. Tween 20™. In one series of embodiments, theFVII polypeptide in formulations I to XVI is polysorbate 80, e.g., Tween80™.

In yet another embodiment, the compositions are selected from the listof:

TABLE 6 Compound Formulation XVII Formulation XVIII Formulation XIXFormulation XX FVII polypeptide 1.0 mg/ml 1.0 mg/ml 1.0 mg/ml 1.0 mg/mlMannitol 25 mg/ml 25 mg/ml 25 mg/ml 25 mg/ml Sucrose 10 mg/ml 10 mg/ml10 mg/ml 10 mg/ml Methionine 0.5 mg/ml — 0.5 mg/ml — Tween 80 0.05 to0.08 0.05 to 0.08 0.05 to 0.08 0.05 to 0.08 mg/ml mg/ml mg/ml mg/ml pH6.0 6.0 5.5 5.5 Compound Formulation XXI Formulation XXII FormulationXXIII Formulation XXIV FVII polypeptide 1.0 mg/ml 1.0 mg/ml 1.0 mg/ml1.0 mg/ml Mannitol 25 mg/ml 25 mg/ml 25 mg/ml 25 mg/ml Sucrose 10 mg/ml10 mg/ml 10 mg/ml 10 mg/ml Methionine 0.5 mg/ml — 0.5 mg/ml — Tween 800.05 to 0.08 0.05 to 0.08 0.07 mg/ml 0.07 mg/ml mg/ml mg/ml pH 6.5 6.55.5 5.5 Compound Formulation XXV Formulation XXVI Formulation XXVIIFormulation XXVIII FVII polypeptide 1.0 mg/ml 1.0 mg/ml 1.0 mg/ml 1.0mg/ml Mannitol 25 mg/ml 25 mg/ml 25 mg/ml 25 mg/ml Sucrose 10 mg/ml 10mg/ml 10 mg/ml 10 mg/ml Methionine 0.5 mg/ml — 0.5 mg/ml — Tween 80 0.07mg/ml 0.07 mg/ml 0.07 mg/ml 0.07 mg/ml pH 6.0 6.0 6.5 6.5

In one series of embodiments, the formulations I to XXVIII furthercomprise one or more components selected from the list of:

-   Ca2+, preferably in an amount of from about 5 to about 15 mM, such    as about 10 mM, and preferably as CaCl2×2H2O;-   NaCl, preferably in an amount of about 50 mM, or about 40 mM, e.g.,    39 mM;-   Histidine, preferably L-Histidine, preferably in an amount of about    10 mM; and-   Glycylglycine, e.g., in an amount of about 10 mM

In another series of embodiments, the compositions contain theexcipients, and amounts thereof, as described in any one of FormulationsI to XXVIII but has a pH of pH 5.5, or 5.6, or 5.7, or, 5.8, or 5.9, or6.1, or 6.2, or 6.3, or 6.4, or 6.5.

In one series of embodiments, the concentration of FVII polypeptide informulations I to XVI is about 1.0 mg/mL.

In one series of embodiments, the FVII polypeptide in formulations I toXXVIII is wild-type human factor VIIa.

In one series of embodiments, the FVII polypeptide in formulations I toXXVIII is a FVII variant.

In different series of embodiments, the FVII polypeptide in formulationsI to XXVIII is selected from the list of: L305V-FVII,L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII,V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII,V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII,V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII,K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII,V158D/M298K-FVII, and S336G-FVII;

In different series of embodiments, the Factor VII polypeptide in anyone of compositions 1 to 48 and 100 to 124 is selected from the list of:From about 0.6 mg/mL to about 10.0 mg/mL, such as from about 0.6 mg/mLto about 8 mg/mL, from about 0.6 mg/mL to about 6 mg/mL, from about 0.6mg/mL to about 5 mg/mL, from about 0.6 mg/mL to about 4 mg/mL, fromabout 0.6 mg/mL to about 3 mg/mL, from about 1.0 mg/mL to about 5 mg/mL,from about 1.0 mg/mL to about 4 mg/mL, from about 1.0 mg/mL to about 3mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9mg/mL, about 1.0 mg/mL, about 1.1 mg/mL, about 1.2 mg/mL, about 1.3mg/mL, about 1.4 mg/mL, about 1.5 mg/mL, about 1.6 mg/mL, about 1.7mg/mL, about 1.8 mg/mL, about 1.9 mg/mL, about 2.0 mg/mL, about 2.1mg/mL, about 2.2 mg/mL, about 2.3 mg/mL, about 2.4 mg/mL, about 2.5mg/mL, about 2.6 mg/mL, about 2.7 mg/mL, about 2.8 mg/mL, about 2.9mg/mL, about 3.0 mg/mL, about 3.1 mg/mL, about 3.2 mg/mL, about 3.3mg/mL, about 3.4 mg/mL, about 3.5 mg/mL, about 3.6 mg/mL, about 3.7mg/mL, about 3.8 mg/mL, about 3.9 mg/mL, and about 4.0 mg/mL.

In another aspect, the invention provides a method of preparing thenovel compositions defined in Tables 3 to 6, comprising the steps of:

-   i) providing a Factor VII polypeptide in a solution comprising at    least one stabilizing agent selected from the group consisting of-   a) a combination of an antioxidant and mannitol;-   b) a combination of methionine and a polyol;-   c) a combination of a saccharide and mannitol;-   d) a combination of sucrose and a polyol; and-   e) methionine-   ii) processing said solution so as to obtain a solid composition    with a moisture content not more than about 3% w/w.

In one embodiment, the polyol is present in an amount ranging from about0.5 to about 75 mg/ml, preferably from about 2 to 45 mg/ml, such as fromabout 5 mg/ml to 45 mg/ml, from about 5 to 35 mg/ml, from about 5 to 25mg/ml, 5 to 20 mg/ml, 20 to 40 mg/ml, or from about 20 to about 30mg/ml,

In one embodiment, the saccharide is present in an amount ranging fromabout 0.5 to 75 mg/ml, preferably from about 2 to 45 mg/ml, such as fromabout 5 mg/ml to 45 mg/ml, from about 5 to 35 mg/ml, from about 5 to 25mg/ml, or from about 5 to 20 mg/ml.

In one embodiment, the antioxidant is in an amount ranging from about0.05 to 10 mg/ml, preferably from about 0.1 to 5 mg/ml, more preferablyfrom about 0.1 mg/ml to 2.5 mg/ml, even more preferably from about 0.1to 2 mg/ml, most preferably from about 0.1 to 1 mg/ml.

In one embodiment, the saccharide is sucrose. In one embodiment, theantioxidant is methionine. In one embodiment, the polyol is mannitol. Inone embodiment, the processing comprises freeze-drying.

The novel compositions of the present invention are reconstituted usingan acceptable, preferably sterile, diluent or carrier, preferably anaqueous carrier. Non-limiting examples of aqueous carriers include Waterfor Injection (WFI) as well as solvents as described in the presentspecification (above) containing at least one of the components selectedfrom the list of: (i) an agent suitable for keeping the pH of saidcomposition in the range of 3 to 9 when dissolved in aqueous solvent inan amount of from about 0.1 mM to 100 mM; and (ii) a tonicity modifyingagent in an amount sufficient to make essentially isotonic thereconstituted solution. In one embodiment, the carrier is WFI; inanother embodiment, the solvent comprises histidine.

EMBODIMENTS OF THE INVENTION Embodiment 1

A kit containing a pharmaceutical medicament, said kit comprising

-   a) a composition comprising a polypeptide and at least one    stabilizing agent, wherein the composition has a moisture content of    not more than about 3%, in a first unit form, and container means    for containing said first unit form; and,-   b) in a second unit form, an administration vehicle comprising a    solvent for reconstitution (solution) of said composition and at    least one of the components selected from the list of:    -   (iii) an agent suitable for keeping the pH of said composition        in the range of 3 to 9 when dissolved in aqueous solvent,        wherein the agent is present in an amount of from about 0.1 mM        to 100 mM,    -   (iv) a tonicity modifying agent in an amount sufficient to make        essentially isotonic the reconstituted solution resulting from        dissolving the composition of the first unit form in the        administration vehicle of the second unit form;-   and container means for containing said second unit form.

Embodiment 2

A kit in accordance with Embodiment 1, wherein the first unit formcomprises at least one component selected from the group of:surfactants, antioxidants, saccharides, and polyols.

Embodiment 3

A kit in accordance with embodiment 2 or embodiment 2, wherein thesecond unit form further comprises at least one component selected fromthe group of: surfactants, antioxidants, saccharides, and polyols.

Embodiment 4

A kit in accordance with any one of embodiments 1 to 3, wherein thepolypeptide is a blood coagulation factor, such as Factor VIII, FactorIX, Factor X, Factor II, Factor V, Factor VII.

Embodiment 5

A kit in accordance with any one of embodiments 1 to 3, wherein thepolypeptide is a vitamin K-dependent polypeptide, such as Factor VII,Factor IX, Factor X, Factor II, Protein C, Protein S, protrombin.

Embodiment 6

A kit in accordance with embodiments 4 or 5, wherein the coagulationfactor polypeptide is selected from the list of: human Factor VIII,human Factor VIIa, a Factor VII-related polypeptide, human Factor IX,human Factor X, activated human Protein C.

Embodiment 7

A kit in accordance with embodiment 6, wherein the FVII-relatedpolypeptide is a factor VII variant selected from the list of:L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII,V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII,V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII,V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII,K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII,V158D/M298K-FVII, and S336G-FVII.

Embodiment 8

A kit in accordance with embodiment 6, wherein the FVII-relatedpolypeptide is a factor VII variant wherein the ratio between theactivity of said Factor VII variant and human factor VIIa (wild-typeFactor VII) is at least 1.25 when tested in one or more of the “In VitroProteolysis Assay” and the “in Vitro Hydrolysis Assay” as described inthe present specification.

Embodiment 9

A kit in accordance with any one of embodiments 1 to 8, wherein the“agent suitable for keeping the pH of said composition in the range of 3to 9 when dissolved in aqueous solvent” is present in an amount of fromabout 0.1 mM to about 50 mM; such as from about 0.1 mM to about 40 mM;from about 0.1 mM to about 35 mM; from about 0.1 mM to about 30 mM; fromabout 0.5 mM to about 25 mM; from about 1 mM to about 20 mM; from about1 mM to about 15 mM; from about 5 mM to about 20 mM; or from about 5 mMto about 15 mM.

Embodiment 10

A kit in accordance with any one of embodiments 1 to 9, wherein the“agent suitable for keeping the pH of said composition in the range of 3to 9 when dissolved in aqueous solvent” is selected from the list of:citric acid, acetic acid, histidine, malic acid, phosphoric acid,tartaric acid, succinic acid, MES, HEPES, imidazole, TRIS, lactic acid,glutaric acid, PIPES and glycylglycine, or a mixture of at least twosuch listed agents, wherein the mixture is able to provide a pH value inthe specified range.

Embodiment 11

A kit in accordance with any one of embodiments 1 to 10, wherein thesecond unit form comprises an agent suitable for keeping the pH of saidcomposition in the range of 4 to 7 when dissolved in aqueous solvent;preferably in the range of 4.5 to 7.5, such as 5 to 7, or 5.5 to 6.5.

Embodiment 12

A kit in accordance with any one of embodiments 1 to 11, wherein thesecond unit form comprises histidine

Embodiment 13

A kit in accordance with any one of embodiments 1 to 12, wherein the“tonicity modifying agent” is selected from the list of: Sodium acetate,sodium lactate, sodium chloride, potassium chloride, calcium chloride,mannitol, glycerol, propylene glycol, or a mixture of at least two suchlisted modifying agents; preferably selected from the list of: sodiumchloride mannitol, glycerol, propylene glycol, calcium chloride, ormixtures thereof.

Embodiment 14

A kit in accordance with embodiment 13, wherein the tonicity modifyingagent comprises Ca2+ or Mg2+.

Embodiment 15

A kit in accordance with any one of embodiments 1 to 14, wherein one orboth of the first and second unit forms further contain a preservative.

Embodiment 16

A kit in accordance with any one of the preceding embodiments; whereinthe first unit form comprises at least one stabilizing agent selectedfrom the group consisting of

-   -   a) a combination of an antioxidant and mannitol;    -   b) a combination of methionine and a polyol;    -   c) a combination of a saccharide and mannitol;    -   d) a combination of sucrose and a polyol;    -   e) methionine; and    -   f) a surfactant        said composition having a moisture content of not more than        about 3%.

Embodiment 17

A kit in accordance with embodiment 16, wherein the combination of anantioxidant and mannitol further comprises a saccharide.

Embodiment 18

A kit in accordance with embodiment 16, wherein the combination ofmethionine and a polyol further comprises a saccharide.

Embodiment 19

A kit in accordance with embodiment 16, wherein the combination of asaccharide and mannitol further comprises an antioxidant.

Embodiment 20

A kit in accordance with embodiment 16, wherein the combination ofsucrose and a polyol further comprises an antioxidant.

Embodiment 21

A kit in accordance with any one of embodiments 16 or 17, wherein thecombination of an antioxidant and mannitol (a) further comprises asurfactant

Embodiment 22

A kit in accordance with any one of embodiments 16 or 18, wherein thecombination of methionine and a polyol (b) further comprises asurfactant.

Embodiment 23

A kit in accordance with any one of embodiments 16 or 19, wherein thecombination of a saccharide and mannitol (c) further comprises asurfactant.

Embodiment 24

A kit in accordance with any one of embodiments 16 or 20, wherein thecombination of sucrose and a polyol (d) further comprises a surfactant.

Embodiment 25

A kit in accordance with embodiment 16, wherein the stabilizing agent isa combination of a surfactant and methionine (e).

Embodiment 26

A kit in accordance with any one of the preceding embodiments, whereinthe antioxidant is selected from the group consisting of homocysteine,cysteine, cystathionine, methionine, gluthatione, and peptidescontaining any one of homocysteine, cysteine, cystathionine, methionineand gluthatione.

Embodiment 27

A kit in accordance with any one of the preceding embodiments, whereinthe saccharide is selected from the group consisting of sucrose,dextrose, lactose, maltose, trehalose, cyclodextrins, maltodextrins anddextrans.

Embodiment 28

A kit in accordance with any one of the preceding embodiments, whereinthe polyol is selected from the group consisting of mannitol, sorbitoland xylitol.

Embodiment 29

A kit in accordance with any one of the preceding embodiments, whereinthe composition of the first unit form is stable such that not more thanabout 5% w/w of the initial content of Factor VII polypeptide isconverted to aggregates upon storage of said composition at 30° C. for 8months, preferably not more than about 4.0% w/w, 3.0% w/w, 2.5% w/w,2.0% w/w, 1.5% w/w, or not more than about 1.0% w/w,

Embodiment 30

A kit in accordance with any one of the preceding embodiments, whereinthe composition of the first unit form is stable such that not more thanabout 6% w/w of the initial content of Factor VII polypeptide isconverted to oxidised forms upon storage of said composition at 30° C.for 8 months, preferably not more than about 5% w/w, 4.0% w/w, 3.0% w/w,2.5% w/w, 2.0% w/w, or not more than about 1.5% w/w.

Embodiment 31

A kit in accordance with any one of the preceding embodiments, whereinsaid polyol is in an amount ranging from about 5% w/w to 90% w/w,preferably from about 18% w/w to 88% w/w, 18% to 83%, 25% to 80%, 40% to80%, 50% to 80%, or from about 50% w/w to 70% w/w.

Embodiment 32

A kit in accordance with any one of the preceding embodiments, whereinsaid saccharide is in an amount ranging from about 0 to 85% w/w,preferably from about 3% w/w to 80% w/w, 7% to 75%, 10% to 70%, 10% to50%, 10% to 40%, or from about 10% w/w to 35% w/w.

Embodiment 33

A kit in accordance with any one of the preceding embodiments, whereinsaid polyol is in a weight ratio relative to said saccharide rangingfrom about 100:1 to 1:50, preferably from about 50:1 to 1:10; 20:1 to1:5; 10:1 to 1:2; 6:1 to 1:2; 4:1 to 1:1; or from about 4:1 to 3:2.

Embodiment 34

A kit in accordance with any one of the preceding embodiments, whereinthe first unit form further comprises a tonicity modifier.

Embodiment 35

A kit in accordance with any one of the preceding embodiments, whereinthe surfactant is selected from the group consisting of polysorbates,such as polysorbate 20 or 80; polyoxyethylene alkyl ethers, such as Brij35®; or poloxamers, such as Poloxamer 188 or 407; and otherethylene/polypropylene block polymers or polyethyleneglycol (PEG) suchas PEG8000.

Embodiment 36

The kit in accordance with any one of the preceding embodiments, whereinthe saccharide is sucrose.

Embodiment 37

The kit in accordance with any one of the preceding embodiments, whereinthe polyol is mannitol.

Embodiment 38

The kit in accordance with any one of the preceding embodiments, whereinFactor VII polypeptide is present in a concentration of from about 0.6mg/ml to about 10.0 mg/ml, such as from about 0.6 mg/ml to about 6mg/ml, from about 0.6 mg/ml to about 5 mg/ml, or from about 0.6 mg/ml toabout 4 mg/ml.

Embodiment 39

The kit in accordance with any one of the preceding embodiments, whereinsaid moisture content is not more than about 2.5% w/w, preferably notmore than about 2% w/w, most preferably not more than about 1.5% w/w

Embodiment 40

The kit in accordance with any one of the preceding embodiments, whereinthe first unit form is a lyophilised cake.

Embodiment 41

The kit in accordance with any one of the preceding embodiments, whereinthe first unit form comprises: Factor VII polypeptide, Mannitol,Sucrose, and a surfactant selected from a polysorbate or a poloxamer,such as Tween 80® or Poloxamer 188®.

Embodiment 42

The kit in accordance with embodiment 41, which further containsmethionine.

Embodiment 43

A kit in accordance with any one of the preceding embodiments 41 or 42,wherein the second unit form contains L-histidine in an amount of fromabout 0.1 mM to about 50 mM; such as from about 0.1 mM to about 40 mM;from about 0.1 mM to about 35 mM; from about 0.1 mM to about 30 mM; fromabout 0.5 mM to about 25 mM; from about 1 mM to about 20 mM; from about1 mM to about 15 mM; from about 5 mM to about 20 mM; or from about 5 mMto about 15 mM.

Embodiment 44

A kit in accordance with any one of the preceding embodiments 41 to 43,wherein the second unit form further contains one or more componentsselected from the list of: CaCl2, NaCl, and Glycylglycine.

Embodiment 45

A method for preparing a liquid formulation of a polypeptide, the methodcomprising the steps of:

-   a) providing a first and a second unit form as described in any one    of embodiments 1 to 44;-   b) mixing said first and second unit forms so as to provide a    dissolved liquid solution of the composition in the administration    vehicle.

Embodiment 46

A method for treating a coagulation factor-responsive syndrome,comprising administering to a subject in need thereof an effectiveamount of a liquid formulation of a coagulation factor prepared by themethod of embodiment 45.

Embodiment 47

A method in accordance with embodiment 46, wherein the coagulationfactor-responsive syndrome is haemophilia, and the coagulation factor isFactor VIII or Factor IX; or the syndrome is sepsis, and the coagulationfactor is protein C or activated protein C.

Embodiment 48

A method in accordance with embodiment 46 for treating a FVII-responsivesyndrome, comprising administering to a subject in need thereof aneffective amount of a liquid formulation of said biological agentprepared by the method of embodiment 45.

Embodiment 49

The method in accordance with embodiment 48, wherein said syndrome isselected from the group consisting of haemophilia A, haemophilia B,Factor XI deficiency, Factor VII deficiency, thrombocytopenia, vonWillebrand's disease, presence of a clotting factor inhibitor, surgery,trauma, dilutional coagulopathy, and anticoagulant therapy.

Embodiment 50

Use of a Factor VII polypeptide for the preparation of a medicament inthe form of a kit as defined in any one of embodiments 1 to 44 fortreatment of a Factor VII-responsive syndrome.

Embodiment 51

A composition comprising a Factor VII polypeptide, and at least onestabilizing agent selected from the group consisting of

-   a) a combination of an antioxidant and mannitol;-   b) a combination of methionine and a polyol;-   c) a combination of a saccharide and mannitol;-   d) a combination of sucrose and a polyol;-   e) methionine; and-   a polysorbate surfactant in an amount of from about 0.06 to 0.08    mg/mL;-   said composition having a moisture content of not more than about    3%;

Embodiment 52

The composition in accordance with embodiment 51, wherein thecombination of an antioxidant and mannitol further comprises asaccharide.

Embodiment 53

The composition in accordance with embodiment 51, wherein thecombination of methionine and a polyol further comprises a saccharide.

Embodiment 54

The composition in accordance with embodiment 51, wherein thecombination of a saccharide and mannitol further comprises anantioxidant.

Embodiment 55

The composition in accordance with embodiment 51, wherein thecombination of sucrose and a polyol further comprises an antioxidant.

Embodiment 56

The composition in accordance with any one of the preceding embodiments51 to 55, wherein the antioxidant is selected from the group consistingof homocysteine, cysteine, cystathionine, methionine, gluthatione, andpeptides containing any one of homocysteine, cysteine, cystathionine,methionine and gluthatione.

Embodiment 57

The composition in accordance with any one of the preceding embodiments51 to 56, wherein the saccharide is selected from the group consistingof sucrose, dextrose, lactose, maltose, trehalose, cyclodextrins,maltodextrins and dextrans.

Embodiment 58

The composition in accordance with any one of the preceding embodiments51 to 57, wherein the polyol is selected from the group consisting ofmannitol, sorbitol and xylitol.

Embodiment 59

The composition in accordance with any one of the preceding embodiments51 to 58, wherein the composition is stable such that not more thanabout 5% w/w of the initial content of Factor VII polypeptide isconverted to aggregates upon storage of said composition at 30° C. for 8months, preferably not more than about 4.0% w/w, 3.0% w/w, 2.5% w/w,2.0% w/w, 1.5% w/w, or not more than about 1.0% w/w,

Embodiment 60

The composition in accordance with any one of the preceding embodiments51 to 59, wherein the composition is stable such that not more thanabout 6% w/w of the initial content of Factor VII polypeptide isconverted to oxidised forms upon storage of said composition at 30° C.for 8 months, preferably not more than about 5% w/w, 4.0% w/w, 3.0% w/w,2.5% w/w, 2.0% w/w, or not more than about 1.5% w/w.

Embodiment 61

The composition in accordance with any one of the preceding embodiments51 to 60, wherein said polyol is in an amount ranging from about 5% w/wto 90% w/w, preferably from about 18% w/w to 88% w/w, 18% to 83%, 25% to80%, 40% to 80%, 50% to 80%, or from about 50% w/w to 70% w/w.

Embodiment 62

The composition in accordance with any one of the preceding embodiments51 to 61, wherein said saccharide is in an amount ranging from about 0to 85% w/w, preferably from about 3% w/w to 80% w/w, 7% to 75%, 10% to70%, 10% to 50%, 10% to 40%, or from about 10% w/w to 35% w/w.

Embodiment 63

The composition in accordance with any one of the preceding embodiments51 to 62, wherein said polyol is in a weight ratio relative to saidsaccharide ranging from about 100:1 to 1:50, preferably from about 50:1to 1:10; 20:1 to 1:5; 10:1 to 1:2; 6:1 to 1:2; 4:1 to 1:1; or from about4:1 to 3:2.

Embodiment 64

The composition in accordance with any one of the preceding embodiments51 to 63, further comprising an agent suitable for keeping the pH ofsaid composition in the range of 3 to 9 when dissolved in aqueoussolvent, preferably the pH is in the range of 4 to 7, more preferred inthe range of 4.5 to 6.5, even more preferred in the range of 5.5 to 6.5.

Embodiment 65

The composition in accordance with embodiment 64, wherein said agent isselected from the group consisting of: citric acid, acetic acid,histidine, malic acid, phosphoric acid, tartaric acid, succinic acid,MES, HEPES, imidazole, TRIS, imidazol, lactic acid, glutaric acid, PIPESand glycylglycine, or a mixture of at least two such listed agents,wherein the mixture is able to provide a pH value in the specifiedrange.

Embodiment 66

The composition in accordance with any one of the preceding embodiments51 to 65, further comprising a tonicity modifier.

Embodiment 67

The composition in accordance with embodiment 66, wherein the tonicitymodifier is selected from the group consisting of: sodium acetate,sodium lactate, sodium chloride, potassium chloride, calcium chloride,mannitol, glycerol, and propylene glycol.

Embodiment 68

The composition in accordance with any one of the preceding embodiments51 to 67, wherein the surfactant is selected from the group consistingof polysorbate 20 or 80, preferably polysorbate 80.

Embodiment 69

The composition in accordance with any one of the preceding embodiments51 to 68, wherein the saccharide is sucrose.

Embodiment 70

The composition in accordance with any one of the preceding embodiments51 to 69, wherein the polyol is mannitol.

Embodiment 71

The composition in accordance with any one of the preceding embodiments51 to 70, wherein the Factor VII Polypeptide is selected from the groupconsisting of Human Factor VIIa, Recombinant Human Factor VIIa and aFactor VII Sequence Variant.

Embodiment 72

The composition in accordance with embodiment 71, wherein the Factor VIIPolypeptide is Human Factor VIIa or Recombinant Human Factor VIIa.

Embodiment 73

The composition in accordance with any one of the preceding embodiments51 to 72, wherein the Factor VII Polypeptide is a Factor VII-relatedpolypeptide wherein the ratio between the activity of said FactorVII-related polypeptide and wild-type Factor VII is at least 1.25 whentested in one or more of the “In Vitro Proteolysis Assay” and the “inVitro Hydrolysis Assay” as described in the present specification.

Embodiment 74

The composition in accordance with any one of the preceding embodiments51 to 73, wherein Factor VII polypeptide is present in a concentrationof from about 0.6 mg/ml to about 10.0 mg/ml, such as from about 0.6mg/ml to about 6 mg/ml, from about 0.6 mg/ml to about 5 mg/ml, or fromabout 0.6 mg/ml to about 4 mg/ml.

Embodiment 75

The composition in accordance with any one of the preceding embodiments51 to 74, wherein said moisture content is not more than about 2.5% w/w,preferably not more than about 2% w/w, most preferably not more thanabout 1.5% w/w

Embodiment 76

The composition in accordance with any one of the preceding embodiments51 to 75, wherein the composition is a lyophilised cake.

Embodiment 77

The composition in accordance with any one of the preceding embodiments,wherein the composition comprises: Factor VII polypeptide, Mannitol,Sucrose, and poloxamer 80, such as Tween

Embodiment 78

The composition in accordance with embodiment 77, which further containsmethionine.

Embodiment 79

The composition in accordance with any one of the preceding embodiments77 to 78, which further contains L-histidine.

Embodiment 80

The composition in accordance with any one of the preceding embodiments77 to 79, which further contains one or more components selected fromthe list of: CaCl2, NaCl, and Glycylglycine.

Embodiments 81

Compositions in accordance with any one of the preceding embodiments 51to 80, selected from the list of:

Compound Formulation I-81 Formulation II-81 FVII polypeptide 0.6 to 10mg/ml 0.6 to 10 mg/ml Mannitol 20 to 40 mg/ml 20 to 40 mg/ml Sucrose 5to 20 mg/ml — Methionine 0-1 mg/ml 0-1 mg/ml Tween 80 0.06 to 0.08 mg/ml0.06 to 0.08 mg/ml pH 5.0 to 7.0 5.0 to 7.0

Embodiment 82

Compositions in accordance with embodiment 81, selected from the listof:

Compound Formulation III-82 Formulation IV-82 FVIIa polypeptide 0.6 to3.0 mg/ml 0.6 to 3.0 mg/ml Mannitol 25 mg/ml 25 mg/ml Sucrose 10 mg/ml10 mg/ml Methionine 0.5 mg/ml — Tween 80 0.06 to 0.08 mg/ml 0.06 to 0.08mg/ml pH 5.5 to 6.5 5.5 to 6.5

Embodiment 83

Compositions in accordance with any one of the preceding embodiments 81or 82, containing polysorbate 80 in an amount of about 0.07 mg/mL.

Embodiment 84

Compositions in accordance with any one of the preceding embodiments 81to 83, containing FVIIa polypeptide in an amount of about 1.0 mg/mL

Embodiment 85

Compositions in accordance with any one of the preceding embodiments 81to 84, further comprising at least one of the components selected fromthe list of: Ca2+ in an amount of about 10 mM, preferably as CaCl2×2H2O;NaCl in an amount of about 50 mM or about 40 mM, e.g., 39 mM; Histidine,preferably L-Histidine in an amount of about 10 mM.

Embodiment 86

Compositions in accordance with any one of the preceding embodiments 81to 85, having a pH of 5.5, or 5.6, or 5.7, or, 5.8, or 5.9, or 6.0, or6.1, or 6.2, or 6.3, or 6.4, or 6.5.

Embodiment 87

A method of preparing the compositions defined in embodiments 51 to 86,comprising the steps of:

-   i) providing a Factor VII polypeptide in a solution comprising at    least one stabilizing agent selected from the group consisting of-   a) a combination of an antioxidant and mannitol;-   b) a combination of methionine and a polyol;-   c) a combination of a saccharide and mannitol;-   d) a combination of sucrose and a polyol; and-   e) methionine; and-   a polysorbate surfactant in an amount of from about 0.06 to 0.08    mg/mL;-   ii) processing said solution so as to obtain a solid composition    with a moisture content not more than about 3% w/w.

Embodiment 88

A method in accordance with embodiment 87, wherein the antioxidant isselected from the group consisting of homocysteine, cysteine,cystathionine, methionine, gluthatione, and peptides containing any oneof homocysteine, cysteine, cystathionine, methionine and gluthatione.

Embodiment 89

A method in accordance with embodiment 87, wherein the saccharide isselected from the group consisting of sucrose, dextrose, lactose,maltose, trehalose, cyclodextrins, maltodextrins and dextrans.

Embodiment 90

A method in accordance with embodiment 87, wherein the polyol isselected from the group consisting of mannitol, sorbitol and xylitol.

Embodiment 91

A method in accordance with embodiment 87, wherein the polyol is presentin an amount ranging from about 0.5 to about 75 mg/ml, preferably fromabout 2 to 45 mg/ml, such as from about 5 mg/ml to 45 mg/ml, from about5 to 35 mg/ml, from about 5 to 25 mg/ml, 5 to 20 mg/ml, 20 to 40 mg/ml,or from about 20 to about 30 mg/ml,

Embodiment 92

A method in accordance with embodiment 87, wherein the saccharide ispresent in an amount ranging from about 0.5 to 75 mg/ml, preferably fromabout 2 to 45 mg/ml, such as from about 5 mg/ml to 45 mg/ml, from about5 to 35 mg/ml, from about 5 to 25 mg/ml, or from about 5 to 20 mg/ml.

Embodiment 93

A method in accordance with embodiment 87, wherein the antioxidant is inan amount ranging from about 0.05 to 10 mg/ml, preferably from about 0.1to 5 mg/ml, more preferably from about 0.1 mg/ml to 2.5 mg/ml, even morepreferably from about 0.1 to 2 mg/ml, most preferably from about 0.1 to1 mg/ml.

Embodiment 94

A method in accordance with embodiment 87, wherein the saccharide issucrose.

Embodiment 95

A method in accordance with embodiment 87, wherein the antioxidant ismethionine.

Embodiment 96

A method in accordance with embodiment 87, wherein the polyol ismannitol.

Embodiment 97

A method in accordance with embodiment 87, wherein the processingcomprises freeze-drying.

Embodiment 98

A method for treating a FVII-responsive syndrome, comprisingadministering to a subject in need thereof an effective amount of acomposition as defined in any one of embodiments 51 to 86.

Embodiment 99

The method in accordance with embodiment 98, wherein said syndrome isselected from the group consisting of haemophilia A, haemophilia B,Factor XI deficiency, Factor VII deficiency, thrombocytopenia, vonWillebrand's disease, presence of a clotting factor inhibitor, surgery,trauma, dilutional coagulopathy, and anticoagulant therapy.

Embodiment 100

Use of Factor VII polypeptide for the preparation of a medicament fortreating a Factor VII-responsive syndrome, said medicament comprising acomposition as defined in any one of embodiments 51 to 86.

Embodiment 101

The use in accordance with embodiment 100, wherein said syndrome isselected from the group consisting of haemophilia A, haemophilia B,Factor XI deficiency, Factor VII deficiency, thrombocytopenia, vonWillebrand's disease, presence of a clotting factor inhibitor, surgery,trauma, and anticoagulant therapy.

The following examples are offered by way of illustration, not by way oflimitation:

EXAMPLES General methods Example 1 Analytical Methods Used inDetermining Stability Indicating Parameters A. Determination of OxidisedForms by Reverse Phase HPLC (RP-HPLC):

HPLC Column: 4.5×250 mm column packed with butylbonded silica with aparticle size of 5 μm and pore size 300 Å. Column temperature: 70° C.Eluent A: water 99.9% v/v and trifluoracetic acid 0.1% v/v. Eluent B:acetonitrile 80% v/v. trifluoracetic acid 0.09% v/v and water 19.91%v/v. The column was eluted with a linear gradient from X % B to (X+13)%B in 30 minutes. Flow rate: 1.0 ml/min. Detection: 214 nm.

The oxidised forms are methionine sulfoxides of Factor VII Polypeptides.For example the two main derivatives of FVII are Met(O)298 FVII andMet(O)306 FVII.

The content of oxidised forms is expressed as the percentage of theinitial amount of Factor VII in the composition upon preparation that isrecovered as oxidised forms of Factor VII.

B. Determination of Aggregates of Factor VII Polypeptides by HighPerformance Gel Permeation Chromatography (GP-HPLC).

GP-HPLC was run on a Waters Polypeptide Pak 300 SW column. 7.5×300 mm.using 0.2 M ammoniumsulfat pH 7.0 containing 5% isopropanol as themobile phase. Flow rate: 0.5 ml/min and detection: 215 nm.

The content of aggregates is expressed as the percentage of the initialamount of Factor VII in the composition upon preparation that isrecovered as dimeric, oligomeric and polymeric forms of Factor VII.

Example 2 Assays for Testing Biological Activity of Factor VIIPolypeptides Test for Factor VIIa Activity:

A suitable assay for testing for Factor VIIa activity and therebyselecting suitable Factor VIIa variants can be performed as a simplepreliminary in vitro test: (the “In Vitro Hydrolysis Assay”).

In Vitro Hydrolysis Assay

Native (wild-type) Factor VIIa and Factor VIIa variant (both hereafterreferred to as “Factor VIIa”) may be assayed for specific activities.They may also be assayed in parallel to directly compare their specificactivities. The assay is carried out in a microtiter plate (MaxiSorp,Nunc, Denmark). The chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide(S-2288, Chromogenix, Sweden), final concentration 1 mM, is added toFactor VIIa (final concentration 100 nM) in 50 mM Hepes, pH 7.4,containing 0.1 M NaCl, 5 mM CaCl₂ and 1 mg/ml bovine serum albumin. Theabsorbance at 405 nm is measured continuously in a SpectraMax™ 340 platereader (Molecular Devices, USA). The absorbance developed during a20-minute incubation, after subtraction of the absorbance in a blankwell containing no enzyme, is used to calculate the ratio between theactivities of variant and wild-type Factor VIIa:

Ratio=(A _(405 nm)Factor VIIa variant)/(A _(405 nm)Factor VIIawild-type).

Based thereon, Factor VIIa variants with an activity comparable to orhigher than native Factor VIIa may be identified, such as, for example,variants where the ratio between the activity of the variant and theactivity of native Factor VII (wild-type FVII) is around, versus above1.0.

The activity of Factor VIIa or Factor VIIa variants may also be measuredusing a physiological substrate such as Factor X, suitably at aconcentration of 100-1000 nM, where the Factor Xa generated is measuredafter the addition of a suitable chromogenic substrate (eg. S-2765)(“the In Vitro Proteolysis Assay”). In addition, the activity assay maybe run at physiological temperature.

In Vitro Proteolysis Assay

Native (wild-type) Factor VIIa and Factor VIIa variant (both hereafterreferred to as “Factor VIIa”) are assayed in parallel to directlycompare their specific activities. The assay is carried out in amicrotiter plate (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nM) andFactor X (0.8 microM) in 100 microL 50 mM Hepes, pH 7.4, containing 0.1M NaCl, 5 mM CaCl2 and 1 mg/ml bovine serum albumin, are incubated for15 min. Factor X cleavage is then stopped by the addition of 50 microL50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 20 mM EDTA and 1 mg/mlbovine serum albumin. The amount of Factor Xa generated is measured byaddition of the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide(S-2765, Chromogenix, Sweden), final concentration 0.5 mM. Theabsorbance at 405 nm is measured continuously in a SpectraMax™ 340 platereader (Molecular Devices, USA). The absorbance developed during 10minutes, after subtraction of the absorbance in a blank well containingno FVIIa, is used to calculate the ratio between the proteolyticactivities of variant and wild-type Factor VIIa:

Ratio=(A405 nm Factor VIIa variant)/(A405 nm Factor VIIa wild-type).

Based thereon, Factor VIIa variants with an activity comparable to orhigher than native Factor VIIa may be identified, such as, for example,variants where the ratio between the activity of the variant and theactivity of native Factor VII (wild-type FVII) is around, versus above1.0.

Thrombin Generation Assay:

The ability of Factor VII or Factor VII-related polypeptides or FactorVIII or Factor VIII-related polypeptides (e.g., variants) to generatethrombin can be measured in an assay comprising all relevant coagulationFactors and inhibitors at physiological concentrations and activatedplatelets (as described on p. 543 in Monroe et al. (1997) Brit. J.Haematol. 99, 542-547 which is hereby incorporated as reference).

Clot Assay:

The activity of the Factor VII polypeptides may also be measured using aone-stage clot assay (assay 4) essentially as described in WO 92/15686or U.S. Pat. No. 5,997,864. Briefly, the sample to be tested is dilutedin 50 mM Tris (pH 7.5), 0.1% BSA and 100 μL is incubated with 100 μL ofFactor VII deficient plasma and 200 μL of thromboplastin C containing 10mM Ca²⁺. Clotting times are measured and compared to a standard curveusing a reference standard or a pool of citrated normal human plasma inserial dilution.

Working Examples Example 3 Stability Data for Compositions of rFVIIaFreeze Dried with and without Histidine, Respectively

It will be seen that it is an advantage to freeze-dry without thepresence of Histidine as less Dimers/Oligomers will be formed.

Compositions

Composition A1 B1 C1 D1 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mLSodium chloride 2.92 mg/mL 2.92 mg/mL 2.92 mg/mL 2.92 mg/mL Calciumchloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine1.32 mg/mL 1.32 mg/mL 1.32 mg/mL 1.32 mg/mL L-Histidine — 1.55 mg/mL —1.55 mg/mL Polysorbate 80 0.07 mg/mL 0.07 mg/mL 0.07 mg/mL 0.07 mg/mLMethionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH5.50 5.50 6.0 6.0 Composition E1 F1 G1 H1 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0mg/mL 1.0 mg/mL Sodium chloride 2.34 mg/mL 2.34 mg/mL 2.34 mg/mL 2.34mg/mL Calcium chloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL 1.47 mg/mLGlycylglycine 1.32 mg/mL 1.32 mg/mL 1.32 mg/mL 1.32 mg/mL L-Histidine —1.55 mg/mL — 1.55 mg/mL Polysorbate 80 0.07 mg/mL 0.07 mg/mL 0.07 mg/mL0.07 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL10 mg/mL pH 5.50 5.50 6.0 6.0 Composition I1 J1 K1 L1 rFVIIa 1.0 mg/mL1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Sodium chloride 2.34 mg/mL 2.34 mg/mL 2.34mg/mL 2.34 mg/mL Calcium chloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL1.47 mg/mL Glycylglycine 1.32 mg/mL 1.32 mg/mL 1.32 mg/mL 1.32 mg/mLL-Histidine — 1.55 mg/mL — 1.55 mg/mL Polysorbate 80 0.1 mg/mL 0.1 mg/mL0.1 mg/mL 0.1 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mLMannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL10 mg/mL 10 mg/mL pH 5.50 5.50 6.0 6.0

For reconstitution of compositions A1, C1, E1, G1, I1, and K1 Histidinesolvens 1.55 mg/mL will be used; for composition B1, D1, F1, H1, J1, andL1 will be used Water for injection (WFI).

Example 4 Stability Data for Compositions of rFVIIa Freeze Dried withand without Histidine, and/or Glycylglycine, and/or NaCl, Respectively

It will be seen that it is an advantage to freeze-dry without thepresence of NaCl as this will decrease the risk of collapse of the cake.

Composition A2 B2 C2 D2 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mLSodium chloride 2.92 mg/mL 2.92 mg/mL 2.92 mg/mL 2.92 mg/mL Calciumchloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine1.32 mg/mL 1.32 mg/mL — — L-Histidine — 1.55 mg/mL — 1.55 mg/mLPolysorbate 80 0.07 mg/mL 0.07 mg/mL 0.07 mg/mL 0.07 mg/mL Methionine0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25 mg/mL 25mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH 5.50 5.505.50 5.50 Composition E2 F2 G2 H2 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL1.0 mg/mL Sodium chloride — — — — Calcium chloride 2H20 1.47 mg/mL 1.47mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine 1.32 mg/mL 1.32 mg/mL — —L-Histidine — 1.55 mg/mL — 1.55 mg/mL Polysorbate 80 0.07 mg/mL 0.07mg/mL 0.07 mg/mL 0.07 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10mg/mL 10 mg/mL 10 mg/mL pH 5.50 5.50 5.50 5.50 Composition I2 J2 K2 L2rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Sodium chloride 2.92mg/mL 2.92 mg/mL 2.92 mg/mL 2.92 mg/mL Calcium chloride 2H20 1.47 mg/mL1.47 mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine 1.32 mg/mL 1.32 mg/mL — —L-Histidine — 1.55 mg/mL — 1.55 mg/mL Polysorbate 80 0.07 mg/mL 0.07mg/mL 0.07 mg/mL 0.07 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10mg/mL 10 mg/mL 10 mg/mL pH 6.0 6.0 6.0 6.0 Composition M2 N2 O2 P2rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL 1.0 mg/mL Sodium chloride — — — —Calcium chloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL 1.47 mg/mLGlycylglycine 1.32 mg/mL 1.32 mg/mL — — L-Histidine — 1.55 mg/mL — 1.55mg/mL Polysorbate 80 0.07 mg/mL 0.07 mg/mL 0.07 mg/mL 0.07 mg/mLMethionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH6.0 6.0 6.0 6.0 Composition Q2 R2 S2 T2 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0mg/mL 1.0 mg/mL Sodium chloride 2.34 mg/mL 2.34 mg/mL 2.34 mg/mL 2.34mg/mL Calcium chloride 2H20 1.47 mg/mL 1.47 mg/mL 1.47 mg/mL 1.47 mg/mLGlycylglycine 1.32 mg/mL 1.32 mg/mL — — L-Histidine — 1.55 mg/mL — 1.55mg/mL Polysorbate 80 0.07 mg/mL 0.07 mg/mL 0.07 mg/mL 0.07 mg/mLMethionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH6.0 6.0 6.0 6.0 Composition U2 V2 X2 Y2 rFVIIa 1.0 mg/mL 1.0 mg/mL 1.0mg/mL 1.0 mg/mL Sodium chloride — — — — Calcium chloride 2H20 1.47 mg/mL1.47 mg/mL 1.47 mg/mL 1.47 mg/mL Glycylglycine 1.32 mg/mL 1.32 mg/mL — —L-Histidine — 1.55 mg/mL — 1.55 mg/mL Polysorbate 80 0.07 mg/mL 0.07mg/mL 0.07 mg/mL 0.07 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10mg/mL 10 mg/mL 10 mg/mL pH 6.0 6.0 6.0 6.0

For reconstitution, Histidine solvens 1.55 mg/mL will be used forComposition A2, I2, and Q2; for composition B2, J2, and R2 will be usedWater for injection (WFI).

For reconstitution, Histidine solvens 1.55 mg/mL will be used forComposition C2, K2, and S2; for composition D2, L2, and T2 will be usedWater for injection (WFI).

For reconstitution, Histidine solvens 1.55 mg/mL with NaCl 2.92 mg/mLwill be used for Composition E2, M2, and U2; for composition F2, N2, andV2 will be used Saline Water 2.92 mg/mL.

For reconstitution, Histidine solvens 1.55 mg/mL with NaCl 2.92 mg/mLwill be used for Composition G2, O2, and X2; for composition H2, P2, andY2 will be used Saline Water 2.92 mg/mL.

Example 5 Manufacturing of Compositions

In general, the compositions were prepared from a purified bulksolution. Excipients were added, and the solution was diluted to thedesired concentration of rFVIIa. The resulting solution was sterilefiltered using a sterilised membrane filter (0.2 micron pore size orequivalent) and filled into sterile glass vials. The vials werefreeze-dried, closed with rubber stoppers, and sealed with aluminiumflip-off type caps.

Example 6 Comparison of Content of Soluble Aggregates Formed inFormulations with and without Addition of L-Histidine BeforeFreeze-Drying

The following formulations were prepared (all concentrations stated inmg/ml):

Formulation no. Ingredient 1 2 3 4 rFVIIa 1.0 1.0 1.0 1.0 NaCl 2.34 2.342.34 2.34 CaCl2, 2H2O 1.47 1.47 1.47 1.47 Glycylglycine 1.32 1.32 1.321.32 Polysorbate 80 0.1 0.1 0.1 0.1 L-Methionine 0.5 0.5 0.5 0.5L-Histidine — — 1.55 1.55 Mannitol 30 25 30 25 Sucrose 8 12 8 12 pH 6.06.0 6.0 6.0

The formulations were prepared as described in example 5 using a fillingvolume of 5.3 ml. After freeze-drying the contents of dimer, oligomer,and polymer forms were measured by GP-HPLC as described in example 3.The results are stated below:

Formulation no. 1 2 3 4 Dimer and oligomer forms (%) 1.8 1.6 2.5 2.7Polymer forms (%) <0.3 <0.3 <0.3 <0.3

The results show that formulations 1 and 2 without addition ofL-histidine had lower contents of dimer and oligomer forms afterfreeze-drying as compared to the corresponding formulations 3 and 4,which contained L-histidine.

Example 7 Stability of a Freeze-Dried rFVIIa Formulation A FormulationContaining

rFVIIa 1.0 mg/ml NaCl 2.34 mg/ml CaCl2, 2H2O 1.47 mg/ml Glycylglycine1.32 mg/ml Polysorbate 80 0.07 mg/ml L-Methionine 0.5 mg/ml Mannitol 25mg/ml Sucrose 10 mg/ml pH 6.0was prepared by as described in example 5 using filling volumes of 1.1ml.

The formulation was placed at 25° C. and 40° C. in darkness. Sampleswere collected according to the tables below. After reconstitution ofthe freeze-dried product in 10 mM L-histidine solvent, the contents ofdimer/oligomer/polymer and oxidised forms were measured as described inexample 3, while the activity was determined by one-stage clot assay asdescribed in example 4.

Storage time at 25° C. (months) Parameter 0 3 6 Dimer/oligomer forms (%)1.6 2.0 2.1 Polymer forms (%) <0.3 <0.3 <0.3 Oxidised forms (%) 1.4 1.31.4 Activity (IU/ml) 58000 54400 n.d. Storage time at 40° C. (months)Parameter 0 1 3 6 Dimer/oligomer forms (%) 1.6 2.4 2.5 2.4 Polymer forms(%) <0.3 0.4 0.4 <0.3 Oxidised forms (%) 1.4 1.2 1.3 1.5 Activity(IU/ml) 58000 53600 53300 n.d. n.d.: not determined

Example 8

The influence of sodium chloride on the visual appearance of the freezedried cake was investigated in a factorial design study. Among thevariable parameters was the content of sodium chloride. The result offour formulations included in the study is shown in the table

Formulation Content of Sodium no. chloride (mg/mL) Visual appearance 1 0Solid homogeneous cake 2 0 Solid homogeneous cake 3 3.50 Partlycollapsed cake 4 3.50 Partly collapsed cake

All Formulations Further Contained:

rFVIIa 1.0 mg/mL Calcium chloride 1.47 mg/mL Glycylglycine 1.32 mg/mLPolysorbate 80 0.1 mg/mL Mannitol 40 mg/mL Sucrose 10 mg/mL

In addition formulations 1 and 2 contained 0.5 mg/mL methionine.

pH was adjusted to:

-   6.0 (formulations 1 and 4)-   5.0 (formulations 2 and 3)

Example 9 Stability Data for Compositions of FVII Polypeptide

It will be seen that the compositions are stable with regard toformation of dimers/oligomers after storage at 25° C. for 6 months.

Compositions

Composition A-9 B-9 C-9 D-9 V158D/E296V/M298Q-FVII 0.6 mg/mL 0.6 mg/mL0.6 mg/mL 0.6 mg/mL Sodium chloride 40 mM 40 mM 40 mM 40 mM (2.34 mg/mL)(2.34 mg/mL) (2.34 mg/mL) (2.34 mg/mL) Calcium chloride 2H20 10 mM 10 mM10 mM 10 mM (1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL)Glycylglycine — — — — L-Histidine 10 mM — 10 mM — (1.55 mg/mL) (1.55mg/mL) Polysorbate 80 0.07 mg/mL 0.07 mg/mL 1.0 mg/mL 1.0 mg/mLMethionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH5.0 5.0 5.0 5.0 Composition E-9 F-9 G-9 H-9 V158D/E296V/M298Q-FVII 0.6mg/mL 0.6 mg/mL 0.6 mg/mL 0.6 mg/mL Sodium chloride — 50 mM — 50 mM(2.92 mg/mL) (2.92 mg/mL) Calcium chloride 2H20 10 mM 10 mM 10 mM 10 mM(1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL) Glycylglycine — — —— L-Histidine 10 mM — 10 mM — (1.55 mg/mL) (1.55 mg/mL) Polysorbate 800.07 mg/mL 0.07 mg/mL 1.0 mg/mL 1.0 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH 5.0 5.0 5.0 5.0

For reconstitution of compositions A-9 to H-9 Water for injection (WFI)will be used.

For reconstitution of compositions B-9, D-9, F-9, and H-9 Histidinesolvens 1.55 mg/mL may also be used.

Compositions containing the same ingredients and amounts of ingredientsas compositions A-9 to H-9 but having a pH of 4.5 were also prepared.Furthermore, compositions containing the same ingredients and amounts ofingredients as compositions A-9 to H-9 but having a pH of 5.5 were alsoprepared.

Composition A-9-1 B-9-1 C-9-1 D-9-1 V158D/M298Q-FVII 0.6 mg/mL 0.6 mg/mL0.6 mg/mL 0.6 mg/mL Sodium chloride 40 mM 40 mM 40 mM 40 mM (2.34 mg/mL)(2.34 mg/mL) (2.34 mg/mL) (2.34 mg/mL) Calcium chloride 2H20 10 mM 10 mM10 mM 10 mM (1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL)Glycylglycine — — — — L-Histidine 10 mM — 10 mM — (1.55 mg/mL) (1.55mg/mL) Polysorbate 80 0.07 mg/mL 0.07 mg/mL 1.0 mg/mL 1.0 mg/mLMethionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH5.0 5.0 5.0 5.0 Composition E-9-1 F-9-1 0.6 G-9-1 H-9-1 V158D/M298Q-FVII0.6 mg/mL 0.6 mg/mL 0.6 mg/mL 0.6 mg/mL Sodium chloride — 50 mM — 50 mM(2.92 mg/mL) (2.92 mg/mL) Calcium chloride 2H20 10 mM 10 mM 10 mM 10 mM(1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL) Glycylglycine — — —— L-Histidine 10 mM — 10 mM — (1.55 mg/mL) (1.55 mg/mL) Polysorbate 800.07 mg/mL 0.07 mg/mL 1.0 mg/mL 1.0 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH 5.0 5.0 5.0 5.0

For reconstitution of compositions A-9-1 to H-9-1 Water for injection(WFI) will be used.

For reconstitution of compositions B-9-1, D-9-1, F-9-1, and H-9-1Histidine solvens 1.55 mg/mL may also be used.

Compositions containing the same ingredients and amounts of ingredientsas compositions A-9-1 to H-9-1 but having a pH of 4.5 were alsoprepared. Furthermore, compositions containing the same ingredients andamounts of ingredients as compositions A-9-1 to H-9-1 but having a pH of5.5 were also prepared.

Composition A-9-2 B-9-2 C-9-2 D-9-2 K337A-FVII 0.6 mg/mL 0.6 mg/mL 0.6mg/mL 0.6 mg/mL Sodium chloride 40 mM 40 mM 40 mM 40 mM (2.34 mg/mL)(2.34 mg/mL) (2.34 mg/mL) (2.34 mg/mL) Calcium chloride 2H20 10 mM 10 mM10 mM 10 mM (1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL)Glycylglycine — — — — L-Histidine 10 mM — 10 mM — (1.55 mg/mL) (1.55mg/mL) Polysorbate 80 0.07 mg/mL 0.07 mg/mL 1.0 mg/mL 1.0 mg/mLMethionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH5.0 5.0 5.0 5.0 Composition E-9-2 F-9-2 G-9-2 H-9-2 K337A-FVII 0.6 mg/mL0.6 mg/mL 0.6 mg/mL 0.6 mg/mL Sodium chloride — 50 mM — 50 mM (2.92mg/mL) (2.92 mg/mL) Calcium chloride 2H20 10 mM 10 mM 10 mM 10 mM (1.47mg/mL) (1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL) Glycylglycine — — — —L-Histidine 10 mM — 10 mM — (1.55 mg/mL) (1.55 mg/mL) Polysorbate 800.07 mg/mL 0.07 mg/mL 1.0 mg/mL 1.0 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH 5.0 5.0 5.0 5.0

For reconstitution of compositions A-9-2 to H-9-2 Water for injection(WFI) will be used.

For reconstitution of compositions B-9-2, D-9-2, F-9-2, and H-9-2Histidine solvens 1.55 mg/mL may also be used.

Compositions containing the same ingredients and amounts of ingredientsas compositions A-9-2 to H-9-2 but having a pH of 4.5 were alsoprepared. Furthermore, compositions containing the same ingredients andamounts of ingredients as compositions A-9-2 to H-9-2 but having a pH of5.5 were also prepared.

Composition A-9-3 B-9-3 C-9-3 D-9-3 M298Q-FVIIa 0.6 mg/mL 0.6 mg/mL 0.6mg/mL 0.6 mg/mL Sodium chloride 40 mM 40 mM 40 mM 40 mM (2.34 mg/mL)(2.34 mg/mL) (2.34 mg/mL) (2.34 mg/mL) Calcium chloride 2H20 10 mM 10 mM10 mM 10 mM (1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL)Glycylglycine — — — — L-Histidine 10 mM — 10 mM — (1.55 mg/mL) (1.55mg/mL) Polysorbate 80 0.07 mg/mL 0.07 mg/mL 1.0 mg/mL 1.0 mg/mLMethionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH5.0 5.0 5.0 5.0 Composition E-9-3 F-9-3 G-9-3 H-9-3 M298Q-FVIIa 0.6mg/mL 0.6 mg/mL 0.6 mg/mL 0.6 mg/mL Sodium chloride — 50 mM — 50 mM(2.92 mg/mL) (2.92 mg/mL) Calcium chloride 2H20 10 mM 10 mM 10 mM 10 mM(1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL) Glycylglycine — — —L-Histidine 10 mM — 10 mM — (1.55 mg/mL) (1.55 mg/mL) Polysorbate 800.07 mg/mL 0.07 mg/mL 1.0 mg/mL 1.0 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL0.5 mg/mL 0.5 mg/mL Mannitol 25 mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL pH 5.0 5.0 5.0 5.0

For reconstitution of compositions A-9-3 to H-9-3 Water for injection(WFI) will be used.

For reconstitution of compositions B-9-3, D-9-3, F-9-3, and H-9-3Histidine solvens 1.55 mg/mL may also be used.

Compositions containing the same ingredients and amounts of ingredientsas compositions A-9-3 to H-9-3 but having a pH of 4.5 were alsoprepared. Furthermore, compositions containing the same ingredients andamounts of ingredients as compositions A-9-3 to H-9-3 but having a pH of5.5 were also prepared.

Composition A-9-4 B-9-4 C-9-4 D-9-4 V158D/E296V/M298Q/K337A- 0.6 mg/mL0.6 mg/mL 0.6 mg/mL 0.6 mg/mL FVIIa Sodium chloride 40 mM 40 mM 40 mM 40mM (2.34 mg/mL) (2.34 mg/mL) (2.34 mg/mL) (2.34 mg/mL) Calcium chloride2H20 10 mM 10 mM 10 mM 10 mM (1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL)(1.47 mg/mL) Glycylglycine — — — — L-Histidine 10 mM — 10 mM — (1.55mg/mL) (1.55 mg/mL) Polysorbate 80 0.07 mg/mL 0.07 mg/mL 1.0 mg/mL 1.0mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10mg/mL pH 5.0 5.0 5.0 5.0 Composition E-9-4 F-9-4 G-9-4 H-9-4V158D/E296V/M298Q/K337A- 0.6 mg/mL 0.6 mg/mL 0.6 mg/mL 0.6 mg/mL FVIIaSodium chloride — 50 mM — 50 mM (2.92 mg/mL) (2.92 mg/mL) Calciumchloride 2H20 10 mM 10 mM 10 mM 10 mM (1.47 mg/mL) (1.47 mg/mL) (1.47mg/mL) (1.47 mg/mL) Glycylglycine — — — — L-Histidine 10 mM — 10 mM —(1.55 mg/mL) (1.55 mg/mL) Polysorbate 80 0.07 mg/mL 0.07 mg/mL 1.0 mg/mL1.0 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10mg/mL pH 5.0 5.0 5.0 5.0

For reconstitution of compositions A-9-4 to H-9-4 Water for injection(WFI) will be used.

For reconstitution of compositions B-9-4, D-9-4, F-9-4, and H-9-4Histidine solvens 1.55 mg/mL may also be used.

Compositions containing the same ingredients and amounts of ingredientsas compositions A-9-4 to H-9-4 but having a pH of 4.5 were alsoprepared. Furthermore, compositions containing the same ingredients andamounts of ingredients as compositions A-9-4 to H-9-4 but having a pH of5.5 were also prepared.

Composition A-9-5 B-9-5 C-9-5 D-9-5 V158D/E296V/M298Q/L305V- 0.6 mg/mL0.6 mg/mL 0.6 mg/mL 0.6 mg/mL FVIIa Sodium chloride 40 mM 40 mM 40 mM 40mM (2.34 mg/mL) (2.34 mg/mL) (2.34 mg/mL) (2.34 mg/mL) Calcium chloride2H20 10 mM 10 mM 10 mM 10 mM (1.47 mg/mL) (1.47 mg/mL) (1.47 mg/mL)(1.47 mg/mL) Glycylglycine — — — — L-Histidine 10 mM — 10 mM — (1.55mg/mL) (1.55 mg/mL) Polysorbate 80 0.07 mg/mL 0.07 mg/mL 1.0 mg/mL 1.0mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10mg/mL pH 5.0 5.0 5.0 5.0 Composition E-9-5 F-9-5 G-9-5 H-9-5V158D/E296V/M298Q/L305V- 0.6 mg/mL 0.6 mg/mL 0.6 mg/mL 0.6 mg/mL FVIIaSodium chloride — 50 mM — 50 mM (2.92 mg/mL) (2.92 mg/mL) Calciumchloride 2H20 10 mM 10 mM 10 mM 10 mM (1.47 mg/mL) (1.47 mg/mL) (1.47mg/mL) (1.47 mg/mL) Glycylglycine — — — — L-Histidine 10 mM — 10 mM —(1.55 mg/mL) (1.55 mg/mL) Polysorbate 80 0.07 mg/mL 0.07 mg/mL 1.0 mg/mL1.0 mg/mL Methionine 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL 0.5 mg/mL Mannitol 25mg/mL 25 mg/mL 25 mg/mL 25 mg/mL Sucrose 10 mg/mL 10 mg/mL 10 mg/mL 10mg/mL pH 5.0 5.0 5.0 5.0

For reconstitution of compositions A-9-5 to H-9-5 Water for injection(WFI) will be used.

For reconstitution of compositions B-9-5, D-9-5, F-9-5, and H-9-5Histidine solvens 1.55 mg/mL may also be used.

Compositions containing the same ingredients and amounts of ingredientsas compositions A-9-5 to H-9-5 but having a pH of 4.5 were alsoprepared. Furthermore, compositions containing the same ingredients andamounts of ingredients as compositions A-9-5 to H-9-5 but having a pH of5.5 were also prepared.

1. A kit containing a pharmaceutical medicament, said kit comprising a)a composition comprising (i) a blood coagulation factor polypeptide and(ii) at least one stabilizing agent selected from the group consistingof: surfactants, antioxidants, saccharides, polyols, and combinations ofany of the foregoing, wherein the composition has a moisture content ofnot more than about 3%, in a first unit form, and container means forcontaining said first unit form; and, b) in a second unit form, anadministration vehicle comprising (i) a solvent for reconstitution ofsaid composition and (ii) at least one component selected from the groupconsisting of: 1) an agent suitable for keeping the pH of saidcomposition in the range of 3 to 9 when dissolved in aqueous solvent,wherein the agent is present in an amount of from about 0.1 mM to 100 mMand 2) a tonicity modifying agent in an amount sufficient to makeessentially isotonic the reconstituted solution resulting fromdissolving the composition of the first unit form in the administrationvehicle of the second unit form; and container means for containing saidsecond unit form.
 2. A kit according to claim 1, wherein the second unitform further comprises (iii) at least one component selected from thegroup of: surfactants, antioxidants, saccharides, and polyols.
 3. A kitaccording to claim 1, wherein the polypeptide is selected from the groupconsisting of: human Factor VIII, human Factor VIIa, human Factor IX,human Factor X, activated human Protein C, and a Factor VII sequencevariant.
 4. A kit according to claim 1, wherein the “agent suitable forkeeping the pH of said composition in the range of 3 to 9 when dissolvedin aqueous solvent” is present at a concentration from about 0.1 mM toabout 50 mM.
 5. A kit according to claim 1, wherein the “agent suitablefor keeping the pH of said composition in the range of 3 to 9 whendissolved in aqueous solvent” is selected from the group consisting of:citric acid, acetic acid, histidine, malic acid, phosphoric acid,tartaric acid, succinic acid, MES, HEPES, imidazole, TRIS, lactic acid,glutaric acid, PIPES, glycylglycine, and combinations of any of theforegoing, wherein the mixture is able to provide a pH between 3 and 9.6. A kit according to claim 1, wherein the “tonicity modifying agent” isselected from the group consisting of: Sodium acetate, sodium lactate,sodium chloride, potassium chloride, calcium chloride, magnesiumchloride, mannitol, glycerol, propylene glycol, and combinations of anyof the foregoing.
 7. A kit according to claim 1, wherein the first unitform comprises at least one stabilizing agent selected from the groupconsisting of a) a combination of an antioxidant and mannitol; b) acombination of methionine and a polyol; c) a combination of a saccharideand mannitol; d) a combination of sucrose and a polyol; e) methionine;f) a surfactant; said composition having a moisture content of not morethan about 3%.
 8. A kit according to claim 1, wherein the antioxidant isselected from the group consisting of homocysteine, cysteine,cystathionine, methionine, gluthatione, and peptides containing any oneof homocysteine, cysteine, cystathionine, methionine and gluthatione;preferably methionine.
 9. A kit according to claim 1, wherein thesaccharide is selected from the group consisting of sucrose, dextrose,lactose, maltose, trehalose, cyclodextrins, maltodextrins and dextrans;preferably sucrose.
 10. A kit according to claim 1, wherein the polyolis selected from the group consisting of mannitol, sorbitol and xylitol;preferably mannitol.
 11. A kit according to claim 1, wherein the firstunit form further comprises a tonicity modifier.
 12. A kit according toclaim 3, wherein Factor VII polypeptide is present in a concentration offrom about 0.6 mg/ml to about 10.0 mg/ml.
 13. A method for preparing aliquid formulation of a blood coagulation factor polypeptide, the methodcomprising the steps of: a) providing a first and a second unit form asdefined in claim 1; b) mixing said first and second unit forms so as toprovide a dissolved liquid solution of the composition in theadministration vehicle.
 14. A method for treating a coagulationfactor-responsive syndrome, comprising administering to a subject inneed thereof an effective amount of a liquid formulation of saidcoagulation factor prepared by the method of claim
 13. 15. A methodaccording to claim 14, wherein the syndrome is a FVII-responsivesyndrome and said coagulation factor is a Factor VII polypeptide. 16.The method according to claim 15, wherein said syndrome is selected fromthe group consisting of: acquired or congenital haemophilia A; acquiredor congenital haemophilia B; Factor XI deficiency; Factor VIIdeficiency; Glanzmann thrombastenia; thrombocytopenia; von Willebrand'sdisease; presence of a clotting factor inhibitor, such as inhibitors tofactors VIII or IX; surgery; trauma; dilutional coagulophathy; andanticoagulant therapy.
 17. A composition comprising (i) a Factor VIIpolypeptide and (ii) at least one stabilizing agent selected from thegroup consisting of a) a combination of an antioxidant and mannitol; b)a combination of methionine and a polyol; c) a combination of asaccharide and mannitol; d) a combination of sucrose and a polyol; e)methionine; and f) a polysorbate surfactant in an amount of from about0.06 to 0.08 mg/mL; said composition having a moisture content of notmore than about 3%.
 18. A composition according to claim 17, selectedfrom the group consisting of: Compound Formulation i Formulation ii FVIIpolypeptide 0.6 to 10 mg/ml 0.6 to 10 mg/ml Mannitol 20 to 40 mg/ml 20to 40 mg/ml Sucrose 5 to 20 mg/ml — Methionine 0-1 mg/ml 0-1 mg/mlPolysorbate 0.06 to 0.08 mg/ml 0.06 to 0.08 mg/ml pH 5.0 to 7.0 5.0 to7.0 Compound Formulation iii Formulation iv FVIIa polypeptide 0.6 to 3.0mg/ml 0.6 to 3.0 mg/mL Mannitol 25 mg/ml 25 mg/ml Sucrose 10 mg/ml 10mg/ml Methionine 0.5 mg/ml — Polysorbate 0.06 to 0.08 mg/ml 0.06 to 0.08mg/ml pH 5.5 to 6.5 5.5 to 6.5 Compound Formulation iii Formulation ivFVIIa polypeptide 0.6 to 3.0 mg/ml 0.6 to 3.0 mg/mL Mannitol 25 mg/ml 25mg/ml Sucrose 10 mg/ml 10 mg/ml Methionine 0.5 mg/ml — Polysorbate 0.06to 0.08 mg/ml 0.06 to 0.08 mg/ml pH 5.0 to 6.0 5.0 to 6.0